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Sample records for primary cultured normal

  1. TRAIL mediates apoptosis in cancerous but not normal primary cultured cells of the human reproductive tract.

    PubMed

    Sadarangani, Anil; Kato, Sumie; Espinoza, Natalia; Lange, Soledad; Llados, Carmen; Espinosa, Marisol; Villalón, Manuel; Lipkowitz, Stanley; Cuello, Mauricio; Owen, Gareth I

    2007-01-01

    Cancer of the reproductive tract encompasses malignancies of the uterine corpus, cervix, ovary, Fallopian tube, among others and accounts for 15% of female cancer mortalities. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis by binding to death receptors and offers a promising cancer treatment. The goal of this study was to investigate and characterize the effect of TRAIL in endometrial cancer cell lines and normal (non-cancerous) epithelial cells of endometrial origin. We also examined the effect of TRAIL in other primary cultured cancers and normal cells of the human female reproductive tract and evaluated if TRAIL mediated apoptosis correlated with death receptors and decoy receptors 1 and 2.Herein, we demonstrate that TRAIL at concentrations which kill cancerous cells, does not mediate apoptosis or alter cell viability in normal human endometrium, ovary, cervix or Fallopian tube. The partial inhibition by a caspase 9 inhibitor and the total inhibition by a caspase 8 inhibitor demonstrates the dependency on the extrinsic apoptotic pathway. The selective mortality does not correlate with the presence of death or decoy receptors. These results suggest that TRAIL may be an effective treatment for endometrial cancer and other female reproductive cancers, with minimal secondary effects on healthy tissue.

  2. Differential effects of vitamin D on normal human prostate epithelial and stromal cells in primary culture.

    PubMed

    Krill, D; Stoner, J; Konety, B R; Becich, M J; Getzenberg, R H

    1999-07-01

    Because epidemiologic evidence has demonstrated that vitamin D may play a role in the etiology of prostate cancer, we tested the inhibitory effect of the biologically active form of vitamin D (1,25-D) on the cell proliferation of human prostate epithelial and stromal cells in a chemically defined situation in the presence and absence of dihydrotestosterone (DHT). We also tested the effect of 1,25-D in castrated rats in the presence and absence of flutamide, an androgen receptor blocker. Prostate stromal and epithelial cells were isolated from freshly collected human prostatectomy specimens, and cell proliferation was measured with the MTT assay. Immunohistochemistry was performed to detect the presence of 1,25-D receptors, androgen receptors, smooth muscle actin, and E-cadherin. For in vivo analysis of 1,25-D, male Sprague-Dawley rats were castrated, then treated with either 1,25-D, 1,25-D with flutamide, or vehicle control. Incubation of primary cultures of prostate epithelial cells with 1,25-D at a concentration of 10(-8) M reduced cell proliferation by 40% of controls. The inhibition of growth by 1,25-D was maintained in the presence of DHT. Conversely, the effect of a similar dose of 1,25-D on stromal cell exposure was increased proliferation. In vivo, 1,25-D increased the prostatic weight of castrated rats that had serum testosterone levels below the detectable limit. The addition of flutamide did not alter this effect. These results confirm that vitamin D may be an effective antiproliferative agent of epithelial cells in prostate cancer therapy and support in vivo studies performed in the normal rat prostate.

  3. Lipid-mediated transfection of normal adult human hepatocytes in primary culture.

    PubMed

    Ourlin, J C; Vilarem, M J; Daujat, M; Harricane, M C; Domergue, J; Joyeux, H; Baulieux, J; Maurel, P

    1997-04-05

    The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.

  4. Measurements of intracellular calcium signals in polarized primary cultures of normal and cystic fibrosis human airway epithelia.

    PubMed

    Ribeiro, Carla M P

    2011-01-01

    The airways are continuously challenged by a variety of stimuli including bacteria, viruses, allergens, and inflammatory factors that act as agonists for G protein-coupled receptors (GPCR). Intracellular calcium (Ca(2+) (i)) mobilization in airway epithelia in response to extracellular stimuli regulates key airway innate defense functions, e.g., Ca(2+)-activated Cl(-) secretion, ciliary beating, mucin secretion, and inflammatory responses. Because Ca(2+) (i) mobilization in response to luminal stimuli is larger in CF vs. normal human airway epithelia, alterations in Ca(2+) (i) signals have been associated with the pathogenesis of CF airway disease. Hence, assessment of Ca(2+) (i) signaling has become an important area of CF research. This chapter will focus on measurements of cytoplasmic and mitochondrial Ca(2+) signals resulting from GPCR activation in polarized primary cultures of normal and CF human bronchial epithelia (HBE).

  5. Magnetic Nanodrug Delivery Through the Mucus Layer of Air-Liquid Interface Cultured Primary Normal Human Tracheobronchial Epithelial Cells

    PubMed Central

    Economou, E. C.; Marinelli, S.; Smith, M. C.; Routt, A. A.; Kravets, V. V.; Chu, H. W.; Spendier, K.; Celinski, Z. J.

    2016-01-01

    Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1–100 nm in diameter were prepared in water. BaNPs and conventional 20–30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles. PMID:27774374

  6. Magnetic Nanodrug Delivery Through the Mucus Layer of Air-Liquid Interface Cultured Primary Normal Human Tracheobronchial Epithelial Cells.

    PubMed

    Economou, E C; Marinelli, S; Smith, M C; Routt, A A; Kravets, V V; Chu, H W; Spendier, K; Celinski, Z J

    2016-09-01

    Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1-100 nm in diameter were prepared in water. BaNPs and conventional 20-30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles.

  7. Guidelines and training initiatives that support communication in cross-cultural primary-care settings: appraising their implementability using Normalization Process Theory.

    PubMed

    de Brún, Tomas; de-Brún, Mary O'Reilly; van Weel-Baumgarten, Evelyn; van Weel, Chris; Dowrick, Christopher; Lionis, Christos; O'Donnell, Catherine A; Burns, Nicola; Mair, Frances S; Saridaki, Aristoula; Papadakaki, Maria; Princz, Christine; van den Muijsenbergh, Maria; MacFarlane, Anne

    2015-08-01

    Guidelines and training initiatives (G/TIs) available to support communication in cross-cultural primary health care consultations are not routinely used. We need to understand more about levers and barriers to their implementation and identify G/TIs likely to be successfully implemented in practice. To report a mapping process used to identify G/TIs and to prospectively appraise their implementability, using Normalization Process Theory (NPT). RESTORE is a 4-year EU FP-7 project. We used purposeful and network sampling to identify experts in statutory and non-statutory agencies across Austria, England, Greece, Ireland, Scotland and the Netherlands who recommended G/TI data from the grey literature. In addition, a peer review of literature was conducted in each country. Resulting data were collated using a standardized Protocol Mapping Document. G/TIs were identified for inclusion by (i) initial elimination of incomplete G/TI material; (ii) application of filtering criteria; and (iii) application of NPT. 20 G/TIs met selection criteria: 8 guidelines and 12 training initiatives. Most G/TIs were identified in the Netherlands (n = 7), followed by Ireland (n = 6) and England (n = 5). Fewer were identified in Scotland (n = 2), and none in Greece or Austria. The majority (n = 13) were generated without the inclusion of migrant service users. All 20 were prospectively appraised for potential implementability by applying NPT. NPT is useful as a means of prospectively testing G/TIs for implementability. Results indicate a need to initiate meaningful engagement of migrants in the development of G/TIs. A European-based professional standard for development and assessment of cross-cultural communication resources is advised. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Cultured normal mammalian tissue and process

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Prewett, Tacey L. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor)

    1993-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cell aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  9. Effects of insulin, dexamethasone and cytokines on {alpha}{sub 1}-acid glycoprotein gene expression in primary cultures of normal rat hepatocytes

    SciTech Connect

    Barraud, B.; Balavoine, S.; Feldmann, G.; Lardeux, B.

    1996-04-01

    While the effects of insulin, dexamethasone and cytokines on {alpha}{sub 1}-acid glycoprotein gene expression have been investigated in various hepatoma cell lines, the individual and combined effects of these components on the expression of this gene have been rarely studied in cultured normal rat hepatocytes. In this cell model, we have shown that mRNA levels of {alpha}{sub 1}-acid glycoprotein were not decreased at least during the first 24 h of culture under basal conditions. During these short-term cultures, the expression of {alpha}{sub 1}-acid glycoprotein in normal hepatocytes showed a high degree of responsiveness to dexamethasone alone (20-fold increase) and to dexamethasone associated with various cytokines (interleukin-1{beta}, interleukin-6 and tumor necrosis factor {alpha}) with a 40 to 100-fold increase depending on the cytokine. Insulin alone did not modify {alpha}{sub 1}-acid glycoprotein mRNA; however, this hormone exerted a positive effect (about 50% increase) in the presence of dexamethasone or dexamethasone with cytokines. These results indicate that the regulation of {alpha}{sub 1}-acid glycoprotein in cultured normal rat hepatocytes presents major differences when compared to reported observations in rat hepatoma cell lines. 49 refs., 2 figs., 2 tabs.

  10. Three Dimensional Primary Hepatocyte Culture

    NASA Technical Reports Server (NTRS)

    Yoffe, Boris

    1998-01-01

    Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

  11. Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.

    PubMed Central

    Bata-Csorgo, Z; Hammerberg, C; Voorhees, J J; Cooper, K D

    1995-01-01

    Flow cytometric analysis of primary ex vivo keratinocyte cultures demonstrated that stem cells, (beta 1 integrin+, keratin 1/keratin 10 [K1/K10-], proliferating cell nuclear antigen [PCNA-] [Bata-Csorgo, Zs., C. Hammerberg, J. J. Voorhees, and K. D. Cooper. 1993. J. Exp. Med. 178:1271-1281]) establish such cultures. This methodology also enabled the quantitation of synchronized recruitment of these cells from G0 into G1 of the cell cycle (PCNA expression), which preceded bright beta 1 integrin expression. (beta 1 integrinbright expression has been shown to be a characteristic feature of keratinocyte stem cells in culture (Jones, P. H., and F. M. Watt. 1993. Cell. 73:713-724). Using the above assay, we determined whether lesional T lymphocytes in psoriasis could be directly responsible for the induction of the stem cell hyperproliferation that is characteristic of this disease. Indeed, CD4+ T lymphocytes, cloned from lesional psoriatic skin and stimulated by immobilized anti-CD3 plus fibronectin, promoted psoriatic uninvolved keratinocyte stem cell proliferation via soluble factors. This induction appeared to be through accelerated recruitment of stem cells from their quiescent state (G0) into cell cycle. By contrast, normal keratinocyte stem cells exhibited no such growth stimulation. Supernatants exhibiting growth induction all contained high levels of GM-CSF and gamma-IFN, low IL-3 and TNF-alpha, and variable IL-4. Only anti-gamma-IFN antibody was able to neutralize growth stimulatory activity of the supernatants on psoriatic uninvolved keratinocyte stem cells. However, because recombinant gamma-IFN alone inhibited growth in this assay, these data suggest that, in psoriasis, gamma-IFN acts cooperatively with other growth factors in the immune induction of cell cycle progression by the normally quiescent stem cell keratinocytes. Images PMID:7529261

  12. The current status of primary hepatocyte culture

    PubMed Central

    Mitaka, Toshihiro

    1998-01-01

    Recently, there have been significant advances toward the development of culture conditions that promote proliferation of primary rodent hepatocytes. There are two major methods for the multiplication of hepatocytes in vitro: one is the use of nicotinamide, the other is the use of a nutrient-rich medium. In the medium containing a high concentration of nicotinamide and a growth factor, primary hepatocytes can proliferate well. In this culture condition small mononucleate cells, which are named small hepatocytes, appear and form colonies. Small hepatocytes have a high potential to proliferate while maintaining hepatic characteristics, and can differentiate into mature ones. On the other hand, combining the nutrient-rich medium with 2% DMSO, the proliferated hepatocytes can recover the hepatic differentiated functions and maintain them for a long time. In this review I describe the culture conditions for the proliferation and differentiation of primary hepatocytes and discuss the small hepatocytes, especially their roles in liver growth. PMID:10319020

  13. PRIMARY CULTURES OF DISSOCIATED SYMPATHETIC NEURONS

    PubMed Central

    Mains, Richard E.; Patterson, Paul H.

    1973-01-01

    Rat sympathetic ganglia were disrupted by mechanical agitation to yield dissociated primary neurons, and the conditions for long-term growth in culture of the isolated neurons were examined. The neurons were grown with or without non-neural cells, simply by the addition or deletion of bicarbonate during growth in culture. Fluorescence histochemistry indicated that the isolated neurons contained catecholamines; incubations with radioactive precursors were used to verify the synthesis and accumulation of both dopamine and norepinephrine. The neurons also produced octopamine using tyramine as precursor, but not with tyrosine as the precursor. In the presence of eserine, older cultures synthesized and stored small amounts of acetylcholine. The cultures did not synthesize and accumulate detectable levels of radioactive γ-aminobutyric acid, 5-hydroxytryptamine, or histamine. PMID:4616046

  14. Dependability of sensitivity tests in primary culture.

    PubMed Central

    Waterworth, P M; Del Piano, M

    1976-01-01

    Primary sensitivity tests were done on 90 specimens of infected urine, and the results were compared with those of secondary tests on pure cultures done by three diffusion methods. There was good correlation between the four methods. In a second study, the reliability of primary tests prepared in the clinical laboratory on specimens of pus was assessed, and the frequency with which a definitive result was obtained with different types of specimen was determined. Recommendations are made for the economic use of these tests. Images PMID:1270599

  15. Molluscan cells in culture: primary cell cultures and cell lines

    PubMed Central

    Yoshino, T. P.; Bickham, U.; Bayne, C. J.

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  16. In vitro methods to culture primary human breast epithelial cells.

    PubMed

    Raouf, Afshin; Sun, Yu Jia

    2013-01-01

    Current evidence suggests that much like leukemia, breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore, knowledge about their nature will provide a platform to develop therapies to eliminate these breast cancer stem cells. This concept highlights the need to understand the mechanisms that regulate the normal functions of the breast stem cells and their immediate progeny as alterations to these same mechanisms can cause these primitive cells to act as cancer stem cells. The study of the primitive cell functions relies on the ability to isolate them from primary sources of breast tissue. This chapter describes processing of discarded tissue from reduction mammoplasty samples as sources of normal primary human breast epithelial cells and describes cell culture systems to grow single-cell suspensions prepared from these reduction samples in vitro.

  17. Primary Sarcopenia in Older People with Normal Nutrition.

    PubMed

    Yadigar, S; Yavuzer, H; Yavuzer, S; Cengiz, M; Yürüyen, M; Döventaş, A; Erdinçler, D S

    2016-03-01

    The aim of this study was to investigate the presence of primary sarcopenia in older patients with normal nutrition and to assess the relationships between the primary sarcopenia with anthropometric measurements. In this prospective clinical cross-sectional study, six-hundred patients who applied to Polyclinic of Geriatrics between dates 2010 and 2011 have been evaluated. The 386 patients who were supposed to have potential secondary sarcopenia were excluded from the study. Age, gender, weight, height, BMI, calf and waist circumference, ongoing medications, additional diseases of the 214 patients included in the study have been surveyed. The sarcopenia criteria of EWSGOP have been applied. Two hundred fourteen cases included in the study were composed of 148 female and 66 male subjects. Mean age was 71.8 ± 2.1 years. Sarcopenia was detected in 105 (49%) subjects while 109 (51%) were normal. Sixty-four female (61%) and 41 (39%) male subjects were sarcopenic. Normal group included 84 female (77%) and 25 male (23%) subjects. Incidence of sarcopenia was found higher in the female patients (p<0.001). No statistically significant difference was detected between sarcopenic and normal groups with respect to age, height, weight, calf circumference and evaluation tests. Waist circumference was higher in the sarcopenic group than the normal group (p=0.02). When both groups were analyzed for BMI; 53 (51%) of the 105 sarcopenic patients had BMI over 30 kg/m2 while 29 (27%) and 23 (22%) patients had BMI of 25-30 kg/m2 and below 25 kg/m2, respectively. Incidence of sarcopenia was significantly higher in the group with BMI over 30 kg/m2 when compared with the groups with BMI of 25-30 kg/m2 and below 25 kg/m2 (p=0.01). Sarcopenia that makes older people physically dependent and decreases their quality of life that receive sufficient nutritional support and are also obese should be comprehensively investigated with respect to presence of sarcopenia.

  18. Reference gene for primary culture of prostate cancer cells.

    PubMed

    Souza, Aline Francielle Damo; Brum, Ilma Simoni; Neto, Brasil Silva; Berger, Milton; Branchini, Gisele

    2013-04-01

    Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.

  19. Insulin Cannot Induce Adipogenic Differentiation in Primary Cardiac Cultures

    PubMed Central

    Parameswaran, Sreejit; Sharma, Rajendra K.

    2016-01-01

    Cardiac tissue contains a heterogeneous population of cardiomyocytes and nonmyocyte population especially fibroblasts. Fibroblast differentiation into adipogenic lineage is important for fat accumulation around the heart which is important in cardiac pathology. The differentiation in fibroblast has been observed both spontaneously and due to increased insulin stimulation. The present study aims to observe the effect of insulin in adipogenic differentiation of cardiac cells present in primary murine cardiomyocyte cultures. Oil Red O (ORO) staining has been used for observing the lipid accumulations formed due to adipogenic differentiation in murine cardiomyocyte cultures. The accumulated lipids were quantified by ORO assay and normalized using protein estimation. The lipid accumulation in cardiac cultures did not increase in presence of insulin. However, addition of other growth factors like insulin-like growth factor 1 and epidermal growth factor promoted adipogenic differentiation even in the presence of insulin and other inhibitory molecules such as vitamins. Lipid accumulation also increased in cells grown in media without insulin after an initial exposure to insulin-containing growth media. The current study adds to the existing knowledge that the insulin by itself cannot induce adipogenic induction in the cardiac cultures. The data have significance in the understanding of cardiovascular health especially in diabetic patients. PMID:27574386

  20. Glucose metabolism by adult hepatocytes in primary culture and by cell lines from rat liver.

    PubMed

    Bissell, D M; Levine, G A; Bissell, M J

    1978-03-01

    The metabolic fate of [U-14C]glucose has been examined in detail in adult rat hepatocytes in primary monolayer culture, as well as in two permanent cell lines--Buffalo rat liver (BRL) and transplantable rat hepatoma (HTC) cells-derived from normal rat liver and from rat hepatoma, respectively. Under defined conditions of incubation, at a glucose concentration of 5.5 mM, the three types of cultured liver cells exhibited pronounced differences in glucose metabolism. Primary cultures, like the intact liver, differed from the cell lines in consuming relatively small amounts of glucose and converting approximately 50% of the total metabolized glucose to lactate. By contrast, the permantent cell lines consumed glucose at a 40-fold greater rate than did primary cultures, converting 80--90% of the carbohydrate to lactate. Oxidative metabolism of glucose carbon also differed among the three types of liver culture. Of the total [U-14C]glucose consumed, primary cultures converted approximately 30% to labeled CO2 per hour, whereas the liver cell lines converted 5--10%. Finally, glucose metabolism in primary culture exhibited adaptation as hepatocytes aged in culture, shifting progressively toward the pattern exhibited by the permanent cell lines. This change occurred over a time course similar to that for other kinds of functional change in hepatocytes in primary culture and thus may be relevant to the general problem of phenotypic alteration in liver cell culture.

  1. Primary Teacher Identity, Commitment and Career in Performative School Cultures

    ERIC Educational Resources Information Center

    Troman, Geoff

    2008-01-01

    The research reported here maps changes in primary teachers' identity, commitment and perspectives and subjective experiences of occupational career in the context of performative primary school cultures. The research aimed to provide in-depth knowledge of performative school culture and teachers' subjective experiences in their work of teaching.…

  2. Examining School Culture in Flemish and Chinese Primary Schools

    ERIC Educational Resources Information Center

    Zhu, Chang; Devos, Geert; Tondeur, Jo

    2014-01-01

    The aim of this research is to gain understanding about school culture characteristics of primary schools in the Flemish and Chinese context. The study was carried out in Flanders (Belgium) and China, involving a total of 44 Flemish schools and 40 Chinese schools. The School Culture Scales were used to measure five school culture dimensions with…

  3. Predicting Organizational Commitment from Organizational Culture in Turkish Primary Schools

    ERIC Educational Resources Information Center

    Ipek, Cemalettin

    2010-01-01

    This study aims to describe organizational culture and commitment and to predict organizational commitment from organizational culture in Turkish primary schools. Organizational Culture Scale (Ipek "1999") and Organizational Commitment Scale (Balay "2000") were used in the data gathering process. The data were collected from…

  4. Predicting Organizational Commitment from Organizational Culture in Turkish Primary Schools

    ERIC Educational Resources Information Center

    Ipek, Cemalettin

    2010-01-01

    This study aims to describe organizational culture and commitment and to predict organizational commitment from organizational culture in Turkish primary schools. Organizational Culture Scale (Ipek "1999") and Organizational Commitment Scale (Balay "2000") were used in the data gathering process. The data were collected from…

  5. Examining School Culture in Flemish and Chinese Primary Schools

    ERIC Educational Resources Information Center

    Zhu, Chang; Devos, Geert; Tondeur, Jo

    2014-01-01

    The aim of this research is to gain understanding about school culture characteristics of primary schools in the Flemish and Chinese context. The study was carried out in Flanders (Belgium) and China, involving a total of 44 Flemish schools and 40 Chinese schools. The School Culture Scales were used to measure five school culture dimensions with…

  6. Teaching Cultural History from Primary Events

    ERIC Educational Resources Information Center

    Carson, Robert N.

    2004-01-01

    This article explores the relationship between specific cultural events such as Galileo's work with the pendulum and a curriculum design that seeks to establish in skeletal form a comprehensive epic narrative about the co-evolution of cultural systems and human consciousness. The article explores some of the challenges and some of the strategies…

  7. Teaching Cultural History from Primary Events

    ERIC Educational Resources Information Center

    Carson, Robert N.

    2004-01-01

    This article explores the relationship between specific cultural events such as Galileo's work with the pendulum and a curriculum design that seeks to establish in skeletal form a comprehensive epic narrative about the co-evolution of cultural systems and human consciousness. The article explores some of the challenges and some of the strategies…

  8. 'The normal' as a culture-related concept: historical considerations.

    PubMed

    Wilson, O M

    1976-01-01

    Three definitions of 'normal' are recognized: the normative or ideal, the average or statistical, and the medical or freedom from illness. We still retain from our historical past the notion that mental or emotional illness bespeaks, if not possession by spirits, at least an irreversible condition. A person who has once sought medical treatment for mental sickness is forever after viewed as suspect, as one who may relapse at any time. The modern norm of reason, as against unreason, reinforces distaste for mental illness. The withdrawal of the Eagleton candidacy in 1972 attests that our society is not yet ready to accept mental illness as treatable and curable. To aspire to the presidency or other high office is probably not 'normal' in the sense of not being 'average', and it is entirely possible that the norm of adaptive behavior is incongruent with ambition and exceptional ability. Should the eligibility requirement for leadership then be 'normal' in the sense of free from illness? Abraham Lincoln was subject to periods of acute depression, Winston Churchill was to a degree manic-depressive, and Woodrow Wilson suffered a series of 'nervous breakdowns' before serving as President. History has vindicated the mental ability and leadership qualities of these men. By modern psychiatric criteria for mental health, many creative scientists and artists would be considered abnormal. On the other hand, the concept of normalcy has recently been used to rationalize political misbehavior. Is it possible, then, to develop a viewpoint of 'normal' that is consistent with our culture, and yet at the same time is broad enough to encompass the uncommon?

  9. Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

    PubMed Central

    2010-01-01

    Introduction Normal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells exhibiting various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear to what extent they retain similar levels of differentiation and heterogeneity as that found within breast tissues. Methods We used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression, as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages. Results Human breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal epithelial phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines are enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Furthermore, although normal human mammary epithelial cell lines exhibit features of bi-potent progenitor cells they are unable to differentiate into mature luminal breast epithelial cells under standard culture conditions. Conclusions As a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral

  10. Increased phase synchronization of spontaneous calcium oscillations in epileptic human versus normal rat astrocyte cultures

    NASA Astrophysics Data System (ADS)

    Balázsi, Gábor; Cornell-Bell, Ann H.; Moss, Frank

    2003-06-01

    Stochastic synchronization analysis is applied to intracellular calcium oscillations in astrocyte cultures prepared from epileptic human temporal lobe. The same methods are applied to astrocyte cultures prepared from normal rat hippocampus. Our results indicate that phase-repulsive coupling in epileptic human astrocyte cultures is stronger, leading to an increased synchronization in epileptic human compared to normal rat astrocyte cultures.

  11. CHANGES IN GENE EXPRESSION DURING DIFFERENTIATION OF CULTURED HUMAN PRIMARY BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Primary airway epithelial cell cultures are a useful tool for the in vitro study of normal bronchial cell differentiation and function, airway disease mechanisms, and pathogens and toxin response. Growth of these cells at an air-liquid interface for several days results in the f...

  12. CHANGES IN GENE EXPRESSION DURING DIFFERENTIATION OF CULTURED HUMAN PRIMARY BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Primary airway epithelial cell cultures are a useful tool for the in vitro study of normal bronchial cell differentiation and function, airway disease mechanisms, and pathogens and toxin response. Growth of these cells at an air-liquid interface for several days results in the f...

  13. Intersections between interprofessional practice, cultural competency and primary healthcare.

    PubMed

    Oelke, Nelly D; Thurston, Wilfreda E; Arthur, Nancy

    2013-09-01

    The concepts of interprofessional collaborative practice (IPCP), cultural competency and primary healthcare (PHC) appear to be linked in theory and practice. This discussion article provides arguments explicating the potential linkages between IPCP and cultural competency. We argue that cultural competency is an important component of IPCP both for relationships with patients and/or communities in which providers work and between team members. Organizational structures also play an important role in facilitating IPCP and cultural competency. The integration of both IPCP and cultural competency has the potential to enhance positive health outcomes. Furthermore, we argue IPCP and cultural competency have important implications for PHC service design, given interprofessional teams are a key component of PHC systems. Linking these concepts in providing PHC services can be essential for impacting outcomes at all levels of primary healthcare, including patient, provider and systems.

  14. Grounding Signs of Culture: Primary Intersubjectivity in Social Semiosis

    ERIC Educational Resources Information Center

    Cowley, Stephen J.; Moodley, Sheshni; Fiori-Cowley, Agnese

    2004-01-01

    The article examines how infants are first permeated by culture. Building on Thibault (2000), semiogenesis is traced to the joint activity of primary intersubjectivity. Using an African example, analysis shows how--at 14 weeks--an infant already uses culturally specific indicators of "what a caregiver wants." Human predispositions and…

  15. Grounding Signs of Culture: Primary Intersubjectivity in Social Semiosis

    ERIC Educational Resources Information Center

    Cowley, Stephen J.; Moodley, Sheshni; Fiori-Cowley, Agnese

    2004-01-01

    The article examines how infants are first permeated by culture. Building on Thibault (2000), semiogenesis is traced to the joint activity of primary intersubjectivity. Using an African example, analysis shows how--at 14 weeks--an infant already uses culturally specific indicators of "what a caregiver wants." Human predispositions and…

  16. Methylmalonate toxicity in primary neuronal cultures.

    PubMed

    McLaughlin, B A; Nelson, D; Silver, I A; Erecinska, M; Chesselet, M F

    1998-09-01

    Several inhibitors of mitochondrial complex II cause neuronal death in vivo and in vitro. The goal of the present work was to characterize in vitro the effects of malonate (a competitive blocker of the complex) which induces neuronal death in a pattern similar to that seen in striatum in Huntington's disease. Exposure of striatal and cortical cultures from embryonic rat brain for 24 h to methylmalonate, a compound which produces malonate intracellularly, led to a dose-dependent cell death. Methylmalonate (10 mM) caused >90% mortality of neurons although cortical cells were unexpectedly more vulnerable. Cell death was attenuated in a medium containing antioxidants. Further characterization revealed that DNA laddering could be detected after 3 h of treatment. Morphological observations (videomicroscopy and Hoechst staining) showed that both necrotic and apoptotic cell death occurred in parallel; apoptosis was more prevalent. A decrease in the ATP/ADP ratio was observed after 3 h of treatment with 10 mM methylmalonate. In striatal cultures it occurred concomitantly with a decline in GABA and a rise in aspartate content and the aspartate/glutamate ratio. Changes in ion concentrations were measured in similar cortical cultures from mouse brain. Neuronal [Na+]i increased while [K+]i and membrane potential decreased after 20 min of continuous incubation in 10 mM methylmalonate. These changes progressed with time, and a rise in [Ca2+]i was also observed after 1 h. The results demonstrate that malonate collapses cellular ion gradients, restoration of which imposes an additional load on the already compromised ATP-generation machinery. An early elevation in [Ca2+]i may trigger an increase in activity of proteases, lipases and endonucleases and production of free radicals and DNA damage which, ultimately, leads to cells death. The data also suggest that maturational and/or extrinsic factors are likely to be critical for the increased vulnerability of striatal neurons to

  17. Primary cell culture of human adenocarcinomas--practical considerations.

    PubMed

    Lerescu, Lucian; Tucureanu, Cătălin; Caraş, Iuliana; Neagu, Stefan; Melinceanu, Laura; Sălăgeanu, Aurora

    2008-01-01

    Cell culture is one of the major tools for oncology research, being an excellent system in which to study the biochemistry and molecular biology associated with individual cancer types and to understand cancer cell physiology. Progress in understanding the biology of any type of carcinoma has been impeded by the inability to culture adequately malignant cells from most epithelial tissues. The ultimate in vitro tumor model would completely reflect the in vivo tumor microenvironment in function and mechanism. Unfortunately, such a model does not currently exist. Homogeneous cell lines that can be continuously propagated on plastic surfaces have been extensively used as a surrogate for tumor environment; however they are very different from the in vivo tumor cells. Model systems involving primary culture represent the situation most closely related to the original tissue although they have a number of disadvantages over cell lines, such as the limited ability to repeat studies with a well characterized culture system that can be used in multiple laboratories. The primary culture may contain many types of stromal and infiltrating cell types potentially complicating the interpretation of data. Yet, their properties better reflect the cellular interactions present in intact tissue. The present article reviews the critical steps in obtaining, routine maintenance and cryopreservation of primary tumor cell cultures, based on information from literature and personal experience on the subject. The article also includes an updated protocol for primary tumor cell isolation and culture.

  18. Gelatin for purification and proliferation of primary keratinocyte culture for use in chronic wounds and burns.

    PubMed

    Rahsaz, Marjan; Geramizadeh, Bita; Kaviani, Maryam; Marzban, Saeed

    2015-04-01

    Human epidermal keratinocytes are currently established as a treatment for burns and wounds and have laboratory applications. Keratinocyte culture contamination by unwanted cells and inhibition of cell proliferation are barriers in primary keratinocyte culture. According to the recent literature, these cells are hard to culture. The present study was conducted to evaluate the efficacy of gelatin-coated surfaces in keratinocyte cultures. After enzymatic isolation of keratinocytes from normal epidermis by trypsin, the cells were cultured on gelatin-coated flasks in serum-free medium. Another group of cells were cultured as a control group without gelatin coating. We showed positive effects of surface coating with gelatin on the primary culture of keratinocytes. Culture of these cells on a gelatincoated surface showed better proliferation with suitable morphology. By using gelatin, adhesion of these cells to the surface was more efficient and without contamination by small round cells. Successful primary culture of keratinocytes on a gelatin-coated surface may provide better yield and optimal number of cells for research and clinical applications.

  19. Relevant principal factors affecting the reproducibility of insect primary culture.

    PubMed

    Ogata, Norichika; Iwabuchi, Kikuo

    2017-02-22

    The primary culture of insect cells often suffers from problems with poor reproducibility in the quality of the final cell preparations. The cellular composition of the explants (cell number and cell types), surgical methods (surgical duration and surgical isolation), and physiological and genetic differences between donors may be critical factors affecting the reproducibility of culture. However, little is known about where biological variation (interindividual differences between donors) ends and technical variation (variance in replication of culture conditions) begins. In this study, we cultured larval fat bodies from the Japanese rhinoceros beetle, Allomyrina dichotoma, and evaluated, using linear mixed models, the effect of interindividual variation between donors on the reproducibility of the culture. We also performed transcriptome analysis of the hemocyte-like cells mainly seen in the cultures using RNA sequencing and ultrastructural analyses of hemocytes using a transmission electron microscope, revealing that the cultured cells have many characteristics of insect hemocytes.

  20. Isolation, Purification, and Culture of Primary Murine Sensory Neurons

    PubMed Central

    Katzenell, Sarah; Cabrera, Jorge R.; North, Brian J.; Leib, David A.

    2017-01-01

    Cultured primary neurons have been of extraordinary value for the study of neuronal anatomy, cell biology, and physiology. While use of neuronal cell lines has ease and utility, there are often caveats that arise due to their mitotic nature. This methods article presents detailed methodology for the preparation, purification, and culture of adult murine sensory neurons for the study of herpes simplex virus lytic and latent infections. While virology is the application for our laboratory, these cultures also have broad utility for neurobiologists and cell biologists. While these primary cultures have been highly informative, the methodology is challenging to many investigators. Through publication of this highly detailed protocol, it is our hope that the use of this culture system can spread in the field to allow more rapid progress in furthering our understanding of neurotropic virus infection. PMID:28808974

  1. Primary and Reversible Pisa Syndrome in Juvenile Normal Pressure Hydrocephalus

    PubMed Central

    Leon-Sarmiento, Fidias E.; Pradilla, Gustavo; del Rosario Zambrano, Maria

    2012-01-01

    Objective To report a case of Pisa syndrome in a patient with idiopathic normal pressure hydrocephalus, who had never been exposed to psychotropic medications. Methods A 26 years-old, Colombian, male patient, was referred because he had cognitive abnormalities, gait disturbances and urinary incontinence. This patient also displayed pleurothotonos. Neurofunctional evaluation of sensory and motor integration at peripheral and central nervous system levels were done. Results Pisa syndrome disappeared after spinal tap drainage with further gait, balance and behavioral improvement. A brainstem-thalamocortical deregulation of the central sensory and motor programming, due to the chaotic enlargement of brain ventricles was thought to be the pathophysiological mechanism underlying this case. Conclusion NPH must not be longer considered as an exclusive geriatric disorder. Further, uncommon movement disorders may appear with this disorder, which should be carefully approached to avoid iatrogenic and deleterious pharmacological interventions. PMID:23794788

  2. Preparation of extracellular matrices produced by cultured and primary fibroblasts

    PubMed Central

    Franco-Barraza, Janusz; Beacham, Dorothy A.; Amatangelo, Michael D.; Cukierman, Edna

    2016-01-01

    Fibroblasts secrete and organize extracellular matrix (ECM), which provides structural support for their adhesion, migration, and tissue organization, besides regulating cellular functions such as growth and survival. Cell-to-matrix interactions are vital for vertebrate development. Disorders in these processes have been associated with fibrosis, developmental malformations, cancer, and other diseases. This unit describes a method for preparing a three-dimensional matrix derived from fibroblastic cells; the matrix is three-dimensional, cell and debris free, and attached to a two-dimensional culture surface. Cell adhesion and spreading are normal on these matrices. This matrix can also be compressed into a two-dimensional matrix and solubilized to study the matrix biochemically. Culturing fibroblasts on traditional two-dimensional (2-D) substrates induces an artificial polarity between lower and upper surfaces of these normally nonpolar cells. Not surprisingly, fibroblast morphology and migration differ once suspended in three-dimensional (3-D) collagen gels (Friedl and Brocker, 2000). However, the molecular composition of collagen gels does not mimic the natural fibroblast (i.e., mesenchymal) microenvironment. Fibroblasts secrete and organize ECM, which provides structural support for their adhesion, migration, and tissue organization, in addition to regulating cellular functions such as growth and survival (Buck and Horwitz, 1987; Hay, 1991; Hynes, 1999; Geiger et al., 2001). Cell-to-matrix interactions are vital for vertebrate development. Disorders in these processes have been associated with fibrosis, developmental malformations, cancer (i.e., desmoplastic tumor microenvironment), and other diseases (Rybinski et al., 2014). This unit describes methods for generating tissue culture surfaces coated with a fibroblast-derived 3-D ECM produced and deposited by both established and primary fibroblasts. The matrices closely resemble in vivo mesenchymal matrices and

  3. Short-term primary culture of epithelial cells derived from human breast tumours.

    PubMed Central

    Speirs, V.; Green, A. R.; Walton, D. S.; Kerin, M. J.; Fox, J. N.; Carleton, P. J.; Desai, S. B.; Atkin, S. L.

    1998-01-01

    As experimental models for breast cancer, most studies rely on established human breast cancer cell lines. However, many of these lines were established over 20 years ago, many from pleural effusions rather than the primary tumour, so the validity of using them as representative models is questionable. This paper describes our experiences, over a 3-year period, in establishing short-term epithelial-cell-enriched preparations from primary breast tumours based on differential centrifugation followed by culture in selective media. Epithelial cells were successfully cultured from 55% of samples, but culture success did not appear to be correlated with tumour histology, stage, grade or node status. Epithelial cell-enriched cultures were immunopositive for broad-spectrum cytokeratin and epithelial membrane antigen (EMA). Positivity for keratin 19 confirmed that the cultures contained tumour-derived cells, which additionally showed significantly higher activity of the reductive pathway of the steroid-converting enzyme 17beta-hydroxysteroid dehydrogenase type I. That the cultures contained tumour and not normal epithelial cells was further substantiated by the complete absence of the calmodulin-like gene NB-1 in tumour-derived cultures; this is only associated with normal breast epithelia. Eighty-five per cent of cultures established from oestrogen receptor (ER)-positive tumours expressed ER in vitro; this was functional in 66% of cultures, although ER-positive phenotype was gradually lost over time. In conclusion, epithelial cells can be isolated and maintained as short-term cultures from primary breast tumours irrespective of histopathological or clinical details, providing a model system with a greater biological and clinical relevance than breast cancer cell lines. Images Figure 1 Figure 2 Figure 5 Figure 7 PMID:9836473

  4. Primary cell culture method for the honeybee Apis mellifera.

    PubMed

    Ju, Hyunhee; Ghil, Sungho

    2015-10-01

    Honeybees are among the most important pollinators in nature, and honeybee-associated products are useful in various areas, including the food industry. However, honeybees may be infected by various types of pathogens. The study of honeybee-associated diseases would greatly benefit from a successful cell culture system, but although some honeybee cell culture techniques have been introduced, these methods have not yet been fully established. Here, we describe a primary cell culture method for the honeybee, Apis mellifera. We isolated, sterilized, and seeded egg cells into non-coated cell culture dishes to generate cell aggregates. After approximately 10 d, aggregates were dissociated and seeded to cell culture dishes. Cell passages were continuously performed, with sub-culturing every 3-4 d. The cells expressed non-adherent phenotypes. Their growth increased with the passage number when they were cultured in growth medium based on L-15 insect medium but not Schneider's insect medium. Finally, polymerase chain reaction confirmed that the cells originated from A. mellifera. Our results suggest that the culturing methods described herein are appropriate for isolating primary cells from honeybee eggs. These methods could thus facilitate the study of honeybee-associated pathogenesis, development, and toxicology.

  5. Epigenetic Modifications as Antidedifferentiation Strategy for Primary Hepatocytes in Culture.

    PubMed

    Bolleyn, Jennifer; Fraczek, Joanna; Rogiers, Vera; Vanhaecke, Tamara

    2015-01-01

    A well-known problem of cultured primary hepatocytes is their rapid dedifferentiation. During the last years, several strategies to counteract this phenomenon have been developed, of which changing the in vitro environment is the most popular one. However, mimicking the in vivo setting in vitro by adding soluble media additives or the restoration of both cell-cell and cell-extracellular matrix contacts is not sufficient and only delays the dedifferentiation process instead of counteracting it. In this chapter, new strategies to prevent the deterioration of the liver-specific phenotype of primary hepatocytes in culture by targeting the (epi)genetic mechanisms that drive hepatocellular gene expression are described.

  6. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

    PubMed

    Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

    2015-08-01

    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

  7. Rapid Selection of Mesenchymal Stem and Progenitor Cells in Primary Prostate Stromal Cultures

    PubMed Central

    Brennen, W. Nathaniel; Kisteman, L. Nelleke; Isaacs, John T.

    2016-01-01

    BACKGROUND Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. METHODS Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (>25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-β measured by ELISA. RESULTS MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-β concentrations. All conditions generated populations with an average cell diameter >15 μm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. CONCLUSIONS Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when

  8. Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata).

    PubMed

    Fukuda, Tomokazu; Kurita, Jun; Saito, Tomomi; Yuasa, Kei; Kurita, Masanobu; Donai, Kenichiro; Nitto, Hiroshi; Soichi, Makoto; Nishimori, Katsuhiko; Uchida, Takafumi; Isogai, Emiko; Onuma, Manabu; Sone, Hideko; Oseko, Norihisa; Inoue-Murayama, Miho

    2012-12-01

    The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

  9. [Thyroid stimulating immunoglobulin bioassay using cultured normal human thyroid cells].

    PubMed

    Ando, M; Yamauchi, K; Tanaka, H; Mori, Y; Takatsuki, K; Yamamoto, M; Matsui, N; Tomita, A

    1985-08-20

    It is currently believed that the thyroid stimulating immunoglobulin (TSI) of Graves' disease is involved in the pathogenesis of hyperthyroidism through the stimulation of the adenylate cyclase-cyclic AMP system. To evaluate this mechanism, TSI in the serum of patients with Graves' disease was determined by its ability to generate cyclic AMP (cAMP) in monolayer cells prepared from a normal thyroid gland. The thyroid tissue was digested with collagenase, and the liberated follicles were collected from the supernatant and cultured for 7 days. One gram of thyroid tissue yielded more than 1 X 10(7) monolayer cells which were stored in aliquots at -80C. Cells (1 approximately 2 X 10(4)/0.28 cm2 microtiter well) were incubated for 4 hours in 0.2 ml Hanks solution poor in NaCl, with various amounts of bovine TSH (bTSH) or 1.5 mg/ml Graves' serum IgG extracted by polyethylene glycol. cAMP accumulated in medium and cells was measured by RIA. Total cAMP (both medium and cells) was about 4 times higher when NaCl was deleted from Hanks solution. Moreover, as more than 90% of the cAMP was released into the medium, it was possible to omit the measurement of cellular cAMP, which requires extraction. The increase in medium cAMP concentration was dependent upon the number of cells, incubation time, and dose of bTSH. Time course and dose response curves in medium cAMP stimulated by IgG from 3 Graves' patients paralleled those of bTSH equivalent units. Accordingly, TSI activity could be expressed in bTSH equivalent units (bTSH microUeq). The assay could detect 1.0 or 3.3 microU/ml of bTSH and was highly reproducible. TSI activity in all of 16 IgGs from normal subjects was under 3.3 bTSH microUeq/ml, while it was greater than 3.3 bTSH microUeq/ml in IgGs from 33 of 37 (89%) untreated patients with Graves disease. Of the 13 patients followed for 2 to 7 months while on antithyroid drugs, 12 had greater than 3.3 bTSH microUeq/ml and, with the exception of one, all showed a decrease in

  10. Where Cultural Games Count: The Voices of Primary Classroom Teachers

    ERIC Educational Resources Information Center

    Nabie, Michael Johnson

    2015-01-01

    This study explored Ghanaian primary school teachers' values and challenges of integrating cultural games in teaching mathematics. Using an In-depth conversational interview, ten (10) certificated teachers' voices on the values and challenges of integrating games were examined. Thematic data analysis was applied to the qualitative data from the…

  11. Creativity and Performativity Policies in Primary School Cultures

    ERIC Educational Resources Information Center

    Troman, Geoff; Jeffrey, Bob; Raggl, Andrea

    2007-01-01

    Cultures of performativity in English primary schools refer to systems and relationships of: target-setting; Ofsted inspections; school league tables constructed from pupil test scores; performance management; performance related pay; threshold assessment; and advanced skills teachers. Systems which demand that teachers "perform" and in…

  12.  Alkaline phosphatase normalization is a biomarker of improved survival in primary sclerosing cholangitis.

    PubMed

    Hilscher, Moira; Enders, Felicity B; Carey, Elizabeth J; Lindor, Keith D; Tabibian, James H

    2016-01-01

     Introduction. Recent studies suggest that serum alkaline phosphatase may represent a prognostic biomarker in patients with primary sclerosing cholangitis. However, this association remains poorly understood. Therefore, the aim of this study was to investigate the prognostic significance and clinical correlates of alkaline phosphatase normalization in primary sclerosing cholangitis. This was a retrospective cohort study of patients with a new diagnosis of primary sclerosing cholangitis made at an academic medical center. The primary endpoint was time to hepatobiliaryneoplasia, liver transplantation, or liver-related death. Secondary endpoints included occurrence of and time to alkaline phosphatase normalization. Patients who did and did not achieve normalization were compared with respect to clinical characteristics and endpoint-free survival, and the association between normalization and the primary endpoint was assessed with univariate and multivariate Cox proportional-hazards analyses. Eighty six patients were included in the study, with a total of 755 patient-years of follow-up. Thirty-eight patients (44%) experienced alkaline phosphatase normalization within 12 months of diagnosis. Alkaline phosphatase normalization was associated with longer primary endpoint-free survival (p = 0.0032) and decreased risk of requiring liver transplantation (p = 0.033). Persistent normalization was associated with even fewer adverse endpoints as well as longer survival. In multivariate analyses, alkaline phosphatase normalization (adjusted hazard ratio 0.21, p = 0.012) and baseline bilirubin (adjusted hazard ratio 4.87, p = 0.029) were the only significant predictors of primary endpoint-free survival. Alkaline phosphatase normalization, particularly if persistent, represents a robust biomarker of improved long-term survival and decreased risk of requiring liver transplantation in patients with primary sclerosing cholangitis.

  13. Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures

    SciTech Connect

    Milam, K.M.; Byard, J.L.

    1985-06-30

    Acetaminophen (APAP) metabolism, cytotoxicity, and genotoxicity were measured in primary cultures of rat hepatocytes. Although 3 mM APAP caused a slight increase in cellular release of lactate dehydrogenase into the culture medium, cellular glutathione concentration (an index of APAP metabolism) was reduced by 50%. APAP at 7 mM was significantly more toxic to these hepatocytes and had a similar but more marked effect on glutathione concentrations. In spite of its cytotoxicity, neither dose of APAP stimulated DNA repair synthesis when monitored by the rate of incorporation of (/sup 3/H)thymidine into DNA following exposure to APAP. Thus, although APAP has been shown to be both hepato- and nephrotoxic in several in vivo and in vitro systems, the reactive toxic metabolite of APAP is not genotoxic in rat primary hepatocyte cultures.

  14. Alpha particle induced DNA damage and repair in normal cultured thyrocytes of different proliferation status.

    PubMed

    Lyckesvärd, Madeleine Nordén; Delle, Ulla; Kahu, Helena; Lindegren, Sture; Jensen, Holger; Bäck, Tom; Swanpalmer, John; Elmroth, Kecke

    2014-07-01

    Childhood exposure to ionizing radiation increases the risk of developing thyroid cancer later in life and this is suggested to be due to higher proliferation of the young thyroid. The interest of using high-LET alpha particles from Astatine-211 ((211)At), concentrated in the thyroid by the same mechanism as (131)I [1], in cancer treatment has increased during recent years because of its high efficiency in inducing biological damage and beneficial dose distribution when compared to low-LET radiation. Most knowledge of the DNA damage response in thyroid is from studies using low-LET irradiation and much less is known of high-LET irradiation. In this paper we investigated the DNA damage response and biological consequences to photons from Cobolt-60 ((60)Co) and alpha particles from (211)At in normal primary thyrocytes of different cell cycle status. For both radiation qualities the intensity levels of γH2AX decreased during the first 24h in both cycling and stationary cultures and complete repair was seen in all cultures but cycling cells exposed to (211)At. Compared to stationary cells alpha particles were more harmful for cycling cultures, an effect also seen at the pChk2 levels. Increasing ratios of micronuclei per cell nuclei were seen up to 1Gy (211)At. We found that primary thyrocytes were much more sensitive to alpha particle exposure compared with low-LET photons. Calculations of the relative biological effectiveness yielded higher RBE for cycling cells compared with stationary cultures at a modest level of damage, clearly demonstrating that cell cycle status influences the relative effectiveness of alpha particles. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. [Characterization of epithelial primary culture from human conjunctiva].

    PubMed

    Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

    2014-01-01

    To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  16. Culture-bound syndromes in Hispanic primary care patients.

    PubMed

    Bayles, Bryan P; Katerndahl, David A

    2009-01-01

    We sought to document Hispanic primary care patients' knowledge and experience of five culture-bound syndromes (CBS), as well as the basic socio-cultural correlates of these disorders. A convenience sample of 100 adult Hispanic patients presenting in an urban South Texas primary care clinic was recruited to complete a brief cross-sectional survey, presented in an oral format. Interviews sought information concerning five culture-bound syndromes--susto, empacho, nervios, mal de ojo, and ataques de nervios. Additional demographic, socio-economic, and acculturation data was collected. Descriptive and bivariate statistics (chi square, Fisher's) were used to assess relationships among variables and experience with each CBS. A multivariate logistic analysis was conducted to determine the possible contributions of age, gender, acculturation, and education to the personal experience of a culture-bound syndrome. Results indicate that 77% of respondents had knowledge of all five syndromes, with 42% reporting having personally experienced at least one CBS. Nervios was the most commonly suffered disorder, being reported by 30 respondents. This was followed, in declining order ofprevalence, by susto, mal de ojo, empacho, and ataques de nervios. Multivariate logistic regression analysis found that higher education beyond high school was associated with a slightly decreased likelihood of reporting having suffered from any culture-bound syndrome. While co-occurrence among these disorders occurred, the patterns of predictors suggest that the co-occurrence is not a reflection of mislabeling of one common syndrome. Knowledge of and experience with culture-bound syndromes is common among Hispanic primary care patients in South Texas. Healthcare providers ought to consider discussing these illnesses in a non-judgmental manner with patients who present with symptoms that are consistent with these syndromes. Future studies, with larger sample sizes, are warranted to elucidate the nature

  17. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture

    PubMed Central

    Li, Xingnan; Nadauld, Lincoln; Ootani, Akifumi; Corney, David C.; Pai, Reetesh K.; Gevaert, Olivier; Cantrell, Michael A.; Rack, Paul G.; Neal, James T.; Chan, Carol W-M.; Yeung, Trevor; Gong, Xue; Yuan, Jenny; Wilhelmy, Julie; Robine, Sylvie; Attardi, Laura D.; Plevritis, Sylvia K.; Hung, Kenneth E.; Chen, Chang-Zheng; Ji, Hanlee P.; Kuo, Calvin J.

    2014-01-01

    The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here, a single air-liquid interface culture method was used without modification to engineer oncogenic mutations into primary epithelial/mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia upon KrasG12D expression and/or p53 loss, and readily generated adenocarcinoma upon in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, KrasG12D and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), and versus more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the Insulin-like growth factor-2 (IGF2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues. PMID:24859528

  18. Cultural significance of primary teeth for caregivers in Northeast Brazil.

    PubMed

    Nations, Marilyn K; Calvasina, Paola Gondim; Martin, Michele N; Dias, Hilma Fontenele

    2008-04-01

    This anthropological study critically evaluates Brazilian caregivers' symbolic production and significance of their malnourished offspring's primary teeth, as well as their own, and describes popular dental practices. From January to June 2004, ethnographic interviews of 27 poor, low-literacy mothers were conducted at a public Malnutrition Treatment Center in Fortaleza, Ceará State. Participant observation of clinical pathways and home environments supplemented the data. Content analysis was performed. Results confirm that primary teeth are imbued with cultural significance in Northeast Brazil. Mothers examine mouths, perceive signs of decay, associate primary with permanent tooth healthiness, identify ethnodental illnesses, seek assistance, and perform rituals with exfoliated teeth. The mother's motivation to care for primary teeth is sparked by her memories of past toothache and attempts to avoid stigma and discrimination. Social determinants, not mothers' beliefs or behaviors, are the most critical obstacles to effective dental care. Legitimizing lay knowledge and empowering caregivers and children can improve oral health in Northeast Brazil.

  19. Establishment of Asian citrus psyllid (Diaphorina citri) primary cultures.

    PubMed

    Marutani-Hert, Mizuri; Hunter, Wayne B; Hall, David G

    2009-01-01

    The Asian citrus psyllid (AsCP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a highly competent vector of the phloem-inhabiting bacterium Candidatus Liberibacter asiaticus associated with the citrus disease huanglongbing (HLB). Commonly referred to as citrus greening disease in the USA, HLB causes reduced fruit yields, quality, and ultimately tree death and is considered the most serious citrus disease. HLB has become a major limiting factor to the production of citrus worldwide. Studies of HLB have been impeded by the fact that C. Liberibacter has not yet been cultured on artificial nutrient media. After being acquired by a psyllid, C. Liberibacter asiaticus is reported to replicate within the psyllid and is retained by the psyllid throughout its life span. We therefore hypothesized that C. Liberibacter asiaticus could be cultured in vitro using psyllid cell cultures as the medium and investigated the establishment of a pure culture for AsCP cells. Several commercially available insect cell culture media along with some media we developed were screened for viability to culture cells from AsCP embryos. Cells from psyllid tissues adhered to the plate and migration was observed within 24 h. Cells were maintained at 20 degrees C. We successfully established primary psyllid cell cultures, referred to as DcHH-1, for D. citri Hert-Hunter-1, with a new media, Hert-Hunter-70.

  20. NITROGEN METABOLISM OF NORMAL AND SARCOMATOUS FIBROBLASTS IN PURE CULTURES

    PubMed Central

    Baker, Lillian E.; Carrel, Alexis

    1928-01-01

    1. Both normal and sarcomatous fibroblasts of the rat utilize many different fragments of the protein molecule for their growth in vitro. Alpha and beta proteoses have approximately equal growth-promoting power. 2. A mixture of peptones, peptides, and amino acids, containing a negligible quantity of proteose, produces a temporary proliferation of normal fibroblasts, and an unlimited multiplication of sarcomatous fibroblasts, provided these substances are derived from liver which contains products of unknown nature that complete the nutritive effect of the protein degradation products. 3. Amino acids contribute to the nutrition of the cells, but are unable without the addition of peptides or polypeptides to support their life. 4. The proteolytic products are more toxic to normal than to sarcomatous fibroblasts. The hypothesis is suggested that the greater acidity produced by the large glycolysis of the sarcomatous cells may account for this difference through altering the speed of action of protein synthetizing enzymes. PMID:19869502

  1. Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures

    PubMed Central

    2012-01-01

    Background There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. To understand astrocyte biology, primary astrocyte cultures are most commonly used because of the direct accessibility of astrocytes in this system. However, this advantage can be hindered by microglial contamination. Although several authors have warned regarding microglial contamination in this system, complete microglial elimination has never been achieved. Methods The number and proliferative potential of contaminating microglia in primary astrocyte cultures were quantitatively assessed by immunocytologic and flow cytometric analyses. To examine the utility of clodronate for microglial elimination, primary astrocyte cultures or MG-5 cells were exposed to liposomal or free clodronate, and then immunocytologic, flow cytometric, and gene expression analyses were performed. The gene expression profiles of microglia-eliminated and microglia-contaminated cultures were compared after interleukin-6 (IL-6) stimulation. Results The percentage of contaminating microglia exceeded 15% and continued to increase because of their high proliferative activity in conventional primary astrocyte cultures. These contaminating microglia were selectively eliminated low concentration of liposomal clodronate. Although primary microglia and MG-5 cells were killed by both liposomal and free clodronate, free clodronate significantly affected the viability of astrocytes. In contrast, liposomal clodronate selectively eliminated microglia without affecting the viability, proliferation or activation of astrocytes. The efficacy of liposomal clodronate was much higher than that of previously reported methods used for decreasing microglial contamination. Furthermore, we observed rapid tumor necrosis factor-α and IL-1b gene induction in conventional primary astrocyte

  2. Malignant Transformation of Mouse Primary Keratinocytes by Harvey Sarcoma Virus and Its Modulation by Surrounding Normal Cells

    NASA Astrophysics Data System (ADS)

    Dotto, Gian Paolo; Weinberg, Robert A.; Ariza, Aurelio

    1988-09-01

    The activated ras oncogene that is present in Harvey sarcoma virus is able to induce malignant transformation of pure cultures of mouse primary keratinocytes. Malignant transformation of these cells is demonstrated by their ability to form carcinomas when grafted back onto syngeneic animals. However, expression of the malignant phenotype by the ras-transformed keratinocytes is drastically inhibited by the presence of normal dermal fibroblasts. This inhibitory effect depends on the ratio of fibroblasts to keratinocytes. It can be observed with mitomycin C-treated growth-arrested dermal fibroblasts and not with other cells, such as normal keratinocytes or established fibroblasts. Thus, a cellular environment approximating normal tissue can suppress tumor formation triggered by a single oncogene.

  3. Cross-cultural aspects of depression management in primary care.

    PubMed

    Hails, Katherine; Brill, Charlotte D; Chang, Trina; Yeung, Albert; Fava, Maurizio; Trinh, Nhi-Ha

    2012-08-01

    Major depressive disorder (MDD) is a prevalent illness in minority populations. Minority patients with MDD are often unrecognized and untreated. This review examines promising interventions to address MDD in primary care settings, where minority groups are more likely to seek care. Since 2010, eleven interventions have been developed to address patient-specific and provider-specific barriers, many of which are adaptations of the collaborative care model. Other promising interventions include cultural tailoring of the collaborative care model, as well as the addition of telepsychiatry, motivational interviewing, cultural consultation, and innovations in interpreting. Overall, collaborative care was found feasible and improved satisfaction and treatment engagement of depressed minority patients in primary care. It remains inconclusive whether these newer intervention models improve MDD treatment outcomes. Future research will be needed to establish the effectiveness of these intervention models in improving the treatment outcomes of minority populations with MDD.

  4. Microbiologic and clinical value of primary broth cultures of wound specimens collected with swabs.

    PubMed Central

    Silletti, R P; Ailey, E; Sun, S; Tang, D

    1997-01-01

    In order to assess the microbiologic and clinical value of primary broth culture of wound specimens collected with swabs and submitted to the laboratory in transport medium, we compared the results of primary agar culture with the results of a corresponding primary broth culture for 344 aerobic specimens and 176 anaerobic specimens. While 8.7% (45 of 520) of the specimens yielded organisms from the primary broth culture that were not recovered from the corresponding primary agar culture, only 5.0% (26 of 520) of the specimens yielded organisms from the primary broth culture other than Staphylococcus epidermidis, viridans group streptococci, and Corynebacterium spp. Moreover, the primary broth culture of only 0.6% (3 of 520) of the specimens yielded organisms not recovered from the primary agar culture that caused a change in the therapy of the patient. Our conclusion is that primary broth cultures are unnecessary for the processing of wound specimens properly collected with swabs. PMID:9230370

  5. Rhamnolipids elicit the same cytotoxic sensitivity between cancer cell and normal cell by reducing surface tension of culture medium.

    PubMed

    Jiang, Lifang; Shen, Chong; Long, Xuwei; Zhang, Guoliang; Meng, Qin

    2014-12-01

    Biosurfactant rhamnolipids have been claimed to show biological activities of inhibiting the proliferation of cancer cells. In this study, the cytotoxicity of rhamnolipids was examined on four cancer cells (HepG2, Caco-2, Hela, MCF-7 cells) and two normal cells (HK-2 cell, primary hepatocyte). Interestingly, both cancer cells and normal cells exhibited similar sensitivities to the addition of rhamnolipids in culture medium, and the cytotoxicity was largely attenuated by the presence of fetal bovine serum (FBS) in culture medium. In correlation of the mono-/di-rhamnolipid cytotoxicity with the surface tension of culture medium, it was found that rhamnolipids triggered cytotoxicity whenever the surface tension of culture medium decreased below 41 mN/m irrespective of the FBS content in culture medium, cell line, or rhamnolipid congener. Similarly, each chemical surfactant (Tween-80, sodium dodecyl sulfate, and sodium dodecyl benzene sulfonate) could cause cytotoxicity on HepG2 cells whenever its addition made the surface tension under 41 mN/m in culture medium with or without the presence of FBS. It seems that rhamnolipids, like chemical surfactants, exhibited cytotoxicity by reducing the surface tension of culture medium rather than by changing its specific molecular structure, which had no selection on tumor cells. This study could offer helps to correct the misleading biological activity of rhamnolipids and to avoid the possible large wastes of time and expenses on developing the applications in antitumor drugs.

  6. Primary Postnatal Dorsal Root Ganglion Culture from Conventionally Slaughtered Calves

    PubMed Central

    Fadda, A.; Bärtschi, M.; Hemphill, A.; Widmer, H. R.; Zurbriggen, A.; Perona, P.; Vidondo, B.; Oevermann, A.

    2016-01-01

    Neurological disorders in ruminants have an important impact on veterinary health, but very few host-specific in vitro models have been established to study diseases affecting the nervous system. Here we describe a primary neuronal dorsal root ganglia (DRG) culture derived from calves after being conventionally slaughtered for food consumption. The study focuses on the in vitro characterization of bovine DRG cell populations by immunofluorescence analysis. The effects of various growth factors on neuron viability, neurite outgrowth and arborisation were evaluated by morphological analysis. Bovine DRG neurons are able to survive for more than 4 weeks in culture. GF supplementation is not required for neuronal survival and neurite outgrowth. However, exogenously added growth factors promote neurite outgrowth. DRG cultures from regularly slaughtered calves represent a promising and sustainable host specific model for the investigation of pain and neurological diseases in bovines. PMID:27936156

  7. Organizational culture in the primary healthcare setting of Cyprus

    PubMed Central

    2013-01-01

    Background The concept of organizational culture is important in understanding the behaviour of individuals in organizations as they manage external demands and internal social changes. Cyprus healthcare system is under restructuring and soon a new healthcare scheme will be implemented starting at the Primary Healthcare (PHC) level. The aim of the study was to investigate the underlying culture encountered in the PHC setting of Cyprus and to identify possible differences in desired and prevailing cultures among healthcare professionals. Methods The population of the study included all general practitioners (GPs) and nursing staff working at the 42 PHC centres throughout the island. The shortened version of the Organizational Culture Profile questionnaire comprising 28 statements on organizational values was used in the study. The instrument was already translated and validated in Greek and cross-cultural adaptation was performed. Participants were required to indicate the organization’s characteristic cultural values orientation along a five-point Likert scale ranging from “Very Much = 1” to “Not at all= 5”. Statistical analysis was performed using SPSS 16.0. Student t-test was used to compare means between two groups of variables whereas for more than two groups analysis of variance (ANOVA) was applied. Results From the total of 306 healthcare professionals, 223 participated in the study (72.9%). The majority of participants were women (75.3%) and mean age was 42.6 ± 10.7 years. Culture dimension “performance orientation” was the desired culture among healthcare professionals (mean: 1.39 ± 0.45). “Supportiveness” and “social responsibility” were the main cultures encountered in PHC (means: 2.37 ± 0.80, 2.38 ± 0.83). Statistical significant differences were identified between desired and prevailing cultures for all culture dimensions (p= 0.000). Conclusions This was the first study performed in Cyprus assessing organizational culture in

  8. Organizational culture in the primary healthcare setting of Cyprus.

    PubMed

    Zachariadou, Theodora; Zannetos, Savvas; Pavlakis, Andreas

    2013-03-24

    The concept of organizational culture is important in understanding the behaviour of individuals in organizations as they manage external demands and internal social changes. Cyprus healthcare system is under restructuring and soon a new healthcare scheme will be implemented starting at the Primary Healthcare (PHC) level. The aim of the study was to investigate the underlying culture encountered in the PHC setting of Cyprus and to identify possible differences in desired and prevailing cultures among healthcare professionals. The population of the study included all general practitioners (GPs) and nursing staff working at the 42 PHC centres throughout the island. The shortened version of the Organizational Culture Profile questionnaire comprising 28 statements on organizational values was used in the study. The instrument was already translated and validated in Greek and cross-cultural adaptation was performed. Participants were required to indicate the organization's characteristic cultural values orientation along a five-point Likert scale ranging from "Very Much = 1" to "Not at all= 5". Statistical analysis was performed using SPSS 16.0. Student t-test was used to compare means between two groups of variables whereas for more than two groups analysis of variance (ANOVA) was applied. From the total of 306 healthcare professionals, 223 participated in the study (72.9%). The majority of participants were women (75.3%) and mean age was 42.6 ± 10.7 years. Culture dimension "performance orientation" was the desired culture among healthcare professionals (mean: 1.39 ± 0.45). "Supportiveness" and "social responsibility" were the main cultures encountered in PHC (means: 2.37 ± 0.80, 2.38 ± 0.83). Statistical significant differences were identified between desired and prevailing cultures for all culture dimensions (p= 0.000). This was the first study performed in Cyprus assessing organizational culture in the PHC setting. In the forthcoming health system reform

  9. Surface markers on human lymphocytes: studies of normal subjects and of patients with primary immunodeficiencies

    PubMed Central

    Aiuti, F.; Lacava, V.; Garofalo, J. A.; D'Amelio, R.; D'Asero, C.

    1973-01-01

    Peripheral blood lymphocytes of twenty normal controls and twelve patients with primary immunodeficiencies were examined for surface membrane Ig and receptors for C3 complement (B cell markers) and for spontaneous rosette formation with sheep erythrocytes (T cell markers). In patients with defects in T cell function no lymphocytes forming spontaneous rosettes were seen. In patients with B cell deficiency they were normal or increased. Lymphocytes with membrane immunoglobulins were normal in patients with T cell defect and absent in patients with severe agammaglobulinaemia. Lymphocytes with receptors for C3 complement were increased in patients with T defect and normal in patients with most other forms of immunodeficiency studied. PMID:4587827

  10. Importance of culturing primary lymphocytes at physiological oxygen levels

    PubMed Central

    Atkuri, Kondala R.; Herzenberg, Leonard A.; Niemi, Anna-Kaisa; Cowan, Tina; Herzenberg, Leonore A.

    2007-01-01

    Although studies with primary lymphocytes are almost always conducted in CO2 incubators maintained at atmospheric oxygen levels (atmosO2; 20%), the physiological oxygen levels (physO2; 5%) that cells encounter in vivo are 2–4 times lower. We show here that culturing primary T cells at atmosO2 significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO2 maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore, we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO2 than at physO2. This apparently paradoxical finding, we suggest, may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO2 than at atmosO2; and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO2 that at atmosO2. Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo, the lower proliferative T cell responses at physO2 likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO2 because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture. PMID:17360561

  11. Effects of Ranolazine on Astrocytes and Neurons in Primary Culture.

    PubMed

    Aldasoro, Martin; Guerra-Ojeda, Sol; Aguirre-Rueda, Diana; Mauricio, M Dolores; Vila, Jose M; Marchio, Patricia; Iradi, Antonio; Aldasoro, Constanza; Jorda, Adrian; Obrador, Elena; Valles, Soraya L

    2016-01-01

    Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10-7, 10-6 and 10-5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents.

  12. Effects of Ranolazine on Astrocytes and Neurons in Primary Culture

    PubMed Central

    Aldasoro, Martin; Guerra-Ojeda, Sol; Aguirre-Rueda, Diana; Mauricio, Mª Dolores; Vila, Jose Mª; Marchio, Patricia; Iradi, Antonio; Aldasoro, Constanza; Jorda, Adrian; Obrador, Elena; Valles, Soraya L.

    2016-01-01

    Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10−7, 10−6 and 10−5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents. PMID:26950436

  13. Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells

    SciTech Connect

    Sorscher, E.J.; Kirk, K.L.; Weaver, M.L.; Jilling, T.; Blalock, J.E.; LeBoeuf, R.D. )

    1991-09-01

    The authors have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropryl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment for 24 hr virtually abolished Cl{sup {minus}} transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br{sup {minus}} uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatments, but not after treatments for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl{sup {minus}} transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl{sup {minus}} permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl{sup {minus}} permeability.

  14. Selective isolation and characterization of primary cells from normal breast and tumors reveal plasticity of adipose derived stem cells.

    PubMed

    Weigand, Annika; Boos, Anja M; Tasbihi, Kereshmeh; Beier, Justus P; Dalton, Paul D; Schrauder, Michael; Horch, Raymund E; Beckmann, Matthias W; Strissel, Pamela L; Strick, Reiner

    2016-03-12

    There is a need to establish more cell lines from breast tumors in contrast to immortalized cell lines from metastatic effusions in order to represent the primary tumor and not principally metastatic biology of breast cancer. This investigation describes the simultaneous isolation, characterization, growth and function of primary mammary epithelial cells (MEC), mesenchymal cells (MES) and adipose derived stem cells (ADSC) from four normal breasts, one inflammatory and one triple-negative ductal breast tumors. A total of 17 cell lines were established and gene expression was analyzed for MEC and MES (n = 42) and ADSC (n = 48) and MUC1, pan-KRT, CD90 and GATA-3 by immunofluorescence. DNA fingerprinting to track cell line identity was performed between original primary tissues and isolates. Functional studies included ADSC differentiation, tumor MES and MEC invasion co-cultured with ADSC-conditioned media (CM) and MES adhesion and growth on 3D-printed scaffolds. Comparative analysis showed higher gene expression of EPCAM, CD49f, CDH1 and KRTs for normal MEC lines; MES lines e.g. Vimentin, CD10, ACTA2 and MMP9; and ADSC lines e.g. CD105, CD90, CDH2 and CDH11. Compared to the mean of all four normal breast cell lines, both breast tumor cell lines demonstrated significantly lower ADSC marker gene expression, but higher expression of mesenchymal and invasion gene markers like SNAI1 and MMP2. When compared with four normal ADSC differentiated lineages, both tumor ADSC showed impaired osteogenic and chondrogenic but enhanced adipogenic differentiation and endothelial-like structures, possibly due to high PDGFRB and CD34. Addressing a functional role for overproduction of adipocytes, we initiated 3D-invasion studies including different cell types from the same patient. CM from ADSC differentiating into adipocytes induced tumor MEC 3D-invasion via EMT and amoeboid phenotypes. Normal MES breast cells adhered and proliferated on 3D-printed scaffolds containing 20 fibers

  15. Culturing primary rat inner medullary collecting duct cells.

    PubMed

    Faust, Dörte; Geelhaar, Andrea; Eisermann, Beate; Eichhorst, Jenny; Wiesner, Burkhard; Rosenthal, Walter; Klussmann, Enno; Klussman, Enno

    2013-06-21

    Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion. Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

  16. Culturing Primary Rat Inner Medullary Collecting Duct Cells

    PubMed Central

    Faust, Dörte; Geelhaar, Andrea; Eisermann, Beate; Eichhorst, Jenny; Wiesner, Burkhard; Rosenthal, Walter; Klussman, Enno

    2013-01-01

    Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion. Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories. PMID:23852264

  17. Effect of primary culture medium type for culture of canine fibroblasts on production of cloned dogs.

    PubMed

    Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Choi, Jin; Kim, Jin Wook; Lee, Tae Hee; Lee, Byeong Chun

    2015-09-01

    Fibroblasts are common source of donor cells for SCNT. It is suggested that donor cells' microenvironment, including the primary culture, affects development of reconstructed embryos. To prove this, canine embryos were cloned with fibroblasts that were cultured in two different primary media (RCMEp vs. Dulbecco's modified Eagle's medium [DMEM]) and in vivo developments were compared with relative amount of stemness, reprogramming, apoptosis gene transcripts, and telomerase activity. Donor cells cultured in RCMEp contained a significantly higher amount of SOX2, NANOG, DPPA2, REXO1, HDAC, DNMT1, MECP2 and telomerase activity than those cultured in DMEM (P < 0.05). In vivo developmental potential of cloned embryos with donor cells cultured in RCMEp had a higher birth rate than that of embryos derived from DMEM (P < 0.05). The culture medium can induce changes in gene expression of donor cells and telomerase activity, and these alterations can also affect in vivo developmental competence of the cloned embryos.

  18. Expression, localisation and functional activation of NFAT-2 in normal human skin, psoriasis, and cultured keratocytes

    PubMed Central

    Al-Daraji, Wael I; Malak, Tamer T.; Prescott, Richard J.; Abdellaoui, Adel; Ali, Mahmud M.; Dabash, Tarek; Zelger, Bettina G.; Zelger, Bernhard

    2009-01-01

    Ciclosporin A (CsA) is widely utilized for the treatment of inflammatory skin diseases such as psoriasis. The therapeutic effects of CsA are thought to be mediated via its immunosuppressive action on infiltrating lymphocytes in skin lesions. CsA and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). As calcineurin and NFAT 1 have been shown to be functionally active in cultured human keratocytes, expression of other NFAT family members such as NFAT-2 and possible functional activation was investigated in human keratocytes. RT-PCR and Western Analysis were used to investigate the presence of NFAT-2 mRNA and protein in human keratocytes. Tissue culture of human keratocytes and immunostaining of cells on coverslips and confocal microscopy were used to assess the degree of nuclear localisation of NFAT-2 in cultured cells. Keratome biopsies were taken from patients with psoriasis (lesional and non-lesional skin) and normal skin and immunohistochemistry was used to assess the NFAT-2 localisation in these biopsies using a well characterized anti-NFAT-2 antibody. The NFAT-2 mRNA and protein expression was demonstrated using RT-PCR and Western blotting. Moreover, the expression of NFAT-2 in normal skin, non-lesional and lesional psoriasis showed a striking basal staining suggesting a role for NFAT-2 in keratocytes proliferation. A range of cell types in the skin express NFAT-2. The expression of NFAT-2 in human keratocytes and response to different agonists provides perhaps a unique opportunity to examine the regulation, subcellular localization and kinetics of translocation of different NFATs in primary cultured human cells. In these experiments the author assessed the expression, localization of NFAT-2 in cultured human keratocytes and measured the degree of nuclear localisaion of NFAT-2 using immunofluorescence

  19. Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes.

    PubMed

    Föllmann, W; Weber, S; Birkner, S

    2000-10-01

    Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon

  20. Preliminary study of spectral features of normal and malignant cell cultures

    NASA Astrophysics Data System (ADS)

    Atif, M.; Farooq, W. A.; Siddiqui, Maqsood A.; Al-Khedhairy, Abdulaziz A.

    2016-04-01

    In this study the fluorescence emission spectra of normal and malignant cell cultures were recorded at an excitation wavelength of 290 nm, corresponding to the higher fluorescence intensity at 350 nm (due to tryptophan) of three malignant cells and normal cells. Similarly, Stokes shift spectra were recorded for normal and malignant cell cultures with a shift, Δλ, of 70 nm. The Stokes shift shows the existence of discriminating features between normal and carcinoma cell lines due to the higher concentration of phenylalanine and tryptophan in carcinoma cell lines which are completely absent in normal cell lines. Hence, both the emission spectra and the Stokes shift spectra showed considerably different spectral features between the normal and malignant cells. The preliminary studies indicate the potential application of fluorescence spectroscopy for cancer detection using the spectral features of biofluorophores.

  1. Toxicity of redox cycling pesticides in primary mesencephalic cultures.

    PubMed

    Bonneh-Barkay, Dafna; Langston, William J; Di Monte, Donato A

    2005-01-01

    A loss of nigrostriatal dopaminergic neurons is the primary neurodegenerative feature of Parkinson's disease. Paraquat, a known redox cycling herbicide, has recently been shown to kill selectively nigrostriatal dopaminergic cells in the mouse model. The purpose of this study was to test the ability of paraquat and other redox cycling pesticides to damage dopaminergic neurons in primary mesencephalic cultures. Addition of paraquat, diquat, or benzyl viologen to mesencephalic cultures induced morphological changes (e.g., dystrophic neuronal processes) consistent with dopaminergic cell injury. The three pesticides also caused cell death as assessed by a reduction of the number of tyrosine hydroxylase-immunoreactive neurons and a dose-dependent decrease in [(3)H]dopamine uptake. Quite interestingly, diquat and benzyl viologen were significantly more toxic than paraquat, probably reflecting their more pronounced ability to trigger redox cycling reactions. The data support a role of redox cycling as a mechanism of dopaminergic cell degeneration and suggest that the property of redox cycling should be taken into consideration when evaluating putative environmental risk factors for Parkinson's disease.

  2. Zinc Modulates Nanosilver-Induced Toxicity in Primary Neuronal Cultures.

    PubMed

    Ziemińska, Elżbieta; Strużyńska, Lidia

    2016-02-01

    Silver nanoparticles (NAg) have recently become one of the most commonly used nanomaterials. Since the ability of nanosilver to enter the brain has been confirmed, there has been a need to investigate mechanisms of its neurotoxicity. We previously showed that primary neuronal cultures treated with nanosilver undergo destabilization of calcium homeostasis via a mechanism involving glutamatergic NMDA receptors. Considering the fact that zinc interacts with these receptors, the aim of the present study was to examine the role of zinc in mechanisms of neuronal cell death in primary cultures. In cells treated with nanosilver, we noted an imbalance between extracellular and intracellular zinc levels. Thus, the influence of zinc deficiency and supplementation on nanosilver-evoked cytotoxicity was investigated by treatment with TPEN (a chelator of zinc ions), or ZnCl(2), respectively. Elimination of zinc leads to complete death of nanosilver-treated CGCs. In contrast, supplementation with ZnCl(2) increases viability of CGCs in a dose-dependent manner. Addition of zinc provided protection against the extra/intracellular calcium imbalance in a manner similar to MK-801, an antagonist of NMDA receptors. Zinc chelation by TPEN decreases the mitochondrial potential and dramatically increases the rate of production of reactive oxygen species. Our results indicate that zinc supplementation positively influences nanosilver-evoked changes in CGCs. This is presumed to be due to an inhibitory effect on NMDA-sensitive calcium channels.

  3. A reporter assay for target validation in primary neuronal cultures.

    PubMed

    Pollio, G; Roncarati, R; Seredenina, T; Terstappen, G C; Caricasole, A

    2008-07-15

    The deposition of beta-amyloid peptides (Abeta42 and Abeta40) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD), and genes modulating their brain levels and neuronal effects could result in future disease modifying therapies. The causal association of candidate targets with AD is of paramount importance in current drug discovery, as a lack of efficacy of many candidate drugs is often due to inadequate validation of their pharmacological target. In Alzheimer's as well as in other neurodegenerative diseases, in vitro target validation is hampered by the difficulty of transfecting primary neuronal cultures and assaying the effects of genes on neuronal viability. Here we describe a rapid, sensitive and simple reporter-based assay for the validation of genes putatively associated with Abeta-mediated neurotoxicity, which can in principle be extended to the validation of targets in the context of other neuronal insults. The assay is suitable for the generation of robust and reproducible data in primary neuronal cultures allowing the dissection at a molecular level of complex pathways activated by the toxic insult in a cellular context that more closely represents the real disease situation.

  4. Aspergillus fumigatus biofilm on primary human sinonasal epithelial culture.

    PubMed

    Singhal, Deepti; Baker, Leonie; Wormald, Peter-John; Tan, Lorwai

    2011-01-01

    Bacterial biofilms have been implicated in chronic rhinosinusitis (CRS). However, direct evidence in support of fungal biofilms in sinus disease is lacking in the literature. This study was designed to develop and characterize an in vitro Aspergillus fumigatus biofilm model on primary human sinonasal epithelial cell culture. Sinonasal biopsy specimens harvested during endoscopic sinus surgery of six CRS patients and three pituitary tumor (control) patients were cultured in Dulbecco's modified Eagle media (DMEM; Invitrogen)/Hams F12 airway media to encourage epithelial cell proliferation. Epithelial cells separated by immunomagnetic beads were seeded in tissue culture-treated Y-shaped microslides. At confluence the primary cultures were inoculated with A. fumigatus spores. Fungus was allowed to germinate and form biofilms under two in vitro conditions: (1) static (no flow through of media) and (2) continuous flow coculture (continuous flow movement of media). At regular intervals cocultures were stained with FUN-1, concanavalin A-alexa fluor 488, and examined by confocal scanning laser microscopy. Comstat software was used to assess biomass and thickness. A. fumigatus formed three-dimensional biofilm structures with parallel-packed, cross-linked hyphae and channels/passages. Metabolically active hyphae showed orange-red fluorescing intravacuolar structures. Extracellular matrix (ECM) between/around the hyphae fluoresced intense green. A. fumigatus biofilms development occurred in five stages: (1) conidial attachment to epithelial cells, (2) hyphal proliferation, (3) ECM production, (4) hyphal parallel packing and cross-linking, and (5) channel/pores formation. Mature biofilms showed basal conidial, middle hyphal, and superficial ECM layers. Biofilms formed under flow conditions displayed more robust and faster growth kinetics when compared with that under static conditions, with a thick, stocky, wrinkly/undulating hyphal growth and extensive ECM production. The

  5. Early loss of mitochondrial inner transmembrane potential in khat-induced cell death of primary normal human oral cells.

    PubMed

    Lukandu, Ochiba M; Bredholt, Therese; Neppelberg, Evelyn; Gjertsen, Bjørn T; Johannessen, Anne C; Vintermyr, Olav K; Costea, Daniela Elena

    2009-09-19

    Previous studies suggest the use of khat, a psychostimulant plant used by millions of people in Middle East and Africa, as risk factor for oral cancer. We previously reported that khat is able to induce adverse affects, as cell cycle arrest and apoptosis, in normal human oral cells cultured in vitro. This study further investigates the more specific role played by mitochondria in khat-induced cell death and the kinetics of the events involved in this process. Exposure of primary normal human oral keratinocytes and fibroblasts to khat extract resulted in a swift and sustained decrease of the mitochondrial inner transmembrane potential occurring within 0.5-1h. Loss of mitochondrial membrane potential preceded all other biochemical and morphologic changes, and was associated with a significant decrease in cell survival. Subsequently, apoptosis-inducing factor was released from mitochondria into cytosol and relocated to nucleus. Cyclosporine A and bongkrekic acid delayed both the loss of mitochondrial inner transmembrane potential and the onset of cell death. This study describes a novel mechanism of khat-induced cell death in primary normal oral keratinocytes and fibroblasts involving an early pivotal effect on mitochondrial function and integrity.

  6. Accumulation of silver nanoparticles by cultured primary brain astrocytes.

    PubMed

    Luther, Eva M; Koehler, Yvonne; Diendorf, Joerg; Epple, Matthias; Dringen, Ralf

    2011-09-16

    Silver nanoparticles (AgNP) are components of various food industry products and are frequently used for medical equipment and materials. Although such particles enter the vertebrate brain, little is known on their biocompatibility for brain cells. To study the consequences of an AgNP exposure of brain cells we have treated astrocyte-rich primary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubation of cultured astrocytes with micromolar concentrations of AgNP for up to 24 h resulted in a time- and concentration-dependent accumulation of silver, but did not compromise the cell viability nor lower the cellular glutathione content. In contrast, the incubation of astrocytes for 4 h with identical amounts of silver as AgNO(3) already severely compromised the cell viability and completely deprived the cells of glutathione. The accumulation of AgNP by astrocytes was proportional to the concentration of AgNP applied and significantly lowered by about 30% in the presence of the endocytosis inhibitors chloroquine or amiloride. Incubation at 4 °C reduced the accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed to AgNP at 37 °C. These data demonstrate that viable cultured brain astrocytes efficiently accumulate PVP-coated AgNP in a temperature-dependent process that most likely involves endocytotic pathways.

  7. Accumulation of silver nanoparticles by cultured primary brain astrocytes

    NASA Astrophysics Data System (ADS)

    Luther, Eva M.; Koehler, Yvonne; Diendorf, Joerg; Epple, Matthias; Dringen, Ralf

    2011-09-01

    Silver nanoparticles (AgNP) are components of various food industry products and are frequently used for medical equipment and materials. Although such particles enter the vertebrate brain, little is known on their biocompatibility for brain cells. To study the consequences of an AgNP exposure of brain cells we have treated astrocyte-rich primary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubation of cultured astrocytes with micromolar concentrations of AgNP for up to 24 h resulted in a time- and concentration-dependent accumulation of silver, but did not compromise the cell viability nor lower the cellular glutathione content. In contrast, the incubation of astrocytes for 4 h with identical amounts of silver as AgNO3 already severely compromised the cell viability and completely deprived the cells of glutathione. The accumulation of AgNP by astrocytes was proportional to the concentration of AgNP applied and significantly lowered by about 30% in the presence of the endocytosis inhibitors chloroquine or amiloride. Incubation at 4 °C reduced the accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed to AgNP at 37 °C. These data demonstrate that viable cultured brain astrocytes efficiently accumulate PVP-coated AgNP in a temperature-dependent process that most likely involves endocytotic pathways.

  8. Optimal 3-D culture of primary articular chondrocytes for use in the Rotating Wall Vessel Bioreactor

    PubMed Central

    Mellor, Liliana F.; Baker, Travis L.; Brown, Raquel J.; Catlin, Lindsey W.; Oxford, Julia Thom

    2014-01-01

    INTRODUCTION Reliable culturing methods for primary articular chondrocytes are essential to study the effects of loading and unloading on joint tissue at the cellular level. Due to the limited proliferation capacity of primary chondrocytes and their tendency to dedifferentiate in conventional culture conditions, long-term culturing conditions of primary chondrocytes can be challenging. The goal of this study was to develop a suspension culturing technique that not only would retain the cellular morphology but also maintain gene expression characteristics of primary articular chondrocytes. METHODS Three-dimensional culturing methods were compared and optimized for primary articular chondrocytes in the rotating wall vessel bioreactor, which changes the mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence. We performed gene expression analysis and morphological characterization of cells cultured in alginate beads, Cytopore-2 microcarriers, primary monolayer culture, and passaged monolayer cultures using reverse transcription-PCR and laser scanning confocal microscopy. RESULTS Primary chondrocytes grown on Cytopore-2 microcarriers maintained the phenotypical morphology and gene expression pattern observed in primary bovine articular chondrocytes, and retained these characteristics for up to 9 days. DISCUSSION Our results provide a novel and alternative culturing technique for primary chondrocytes suitable for studies that require suspension such as those using the rotating wall vessel bioreactor. In addition, we provide an alternative culturing technique for primary chondrocytes that can impact future mechanistic studies of osteoarthritis progression, treatments for cartilage damage and repair, and cartilage tissue engineering. PMID:25199120

  9. (-) deprenyl attenuates aluminium induced neurotoxicity in primary cortical cultures.

    PubMed

    Munirathinam, S; Lakshmana, M K; Raju, T R

    1996-06-01

    The role of (-) deprenyl in offering neuroprotection to cortical neurons exposed to Aluminium chloride (AlCl3) was examined. Primary cortical cultures derived from newborn rats were exposed to AlCl3 on 6th day in vitro, at 100,200,400,600,800 and 1000 microM concentrations of AlCl3. After 48 h of AlCl3 exposure, many nerve cell bodies were swollen; a beading of neurites and a disruption of the neuritic network were also observed suggesting neurodegeneration. Lactate dehydrogenase (LDH) efflux increased in a dose-dependent manner (59-120%). (-) Deprenyl co-exposure at concentrations of 10(-7), 10(-8) and 10(-9) M significantly attenuated both the morphological alterations and the LDH efflux induced by AlCl3. This in vitro study has demonstrated that (-) deprenyl can protect neurons from aluminium induced neurotoxicity.

  10. Assessment of cell viability in primary neuronal cultures.

    PubMed

    Aras, Mandar A; Hartnett, Karen A; Aizenman, Elias

    2008-07-01

    This unit contains five protocols for assaying cell viability in vitro using primary neuronal cultures, including a novel method for use with transfected neurons. Three of the assays are based on the principle that cell death cascades alter membrane permeability. The lactate dehydrogenase (LDH) release assay measures the amount of the cytoplasmic enzyme released into the bathing medium, while the trypan blue and propidium iodide assays measure the ability of cells to exclude dye from their cytoplasm. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay measures the mitochondrial activity of viable cells by quantifying the conversion of the tetrazolium salt to its formazan product. Finally, the fifth assay details the measurement of luciferase expression as an indication of neuronal viability within a relatively small population of transfected neurons.

  11. Insulin binding properties of normal and transformed human epidermal cultured keratinocytes

    SciTech Connect

    Verrando, P.; Ortonne, J.P.

    1985-10-01

    Insulin binding to its receptors was studied in cultured normal and transformed (A431 line) human epidermal keratinocytes. The specific binding was a temperature-dependent, saturable process. Normal keratinocytes possess a mean value of about 80,000 receptors per cell. Fifteen hours exposure of the cells to insulin lowered their receptor number (about 65% loss in available sites); these reappeared when the hormone was removed from the culture medium. In the A431 epidermoid carcinoma cell line, there is a net decrease in insulin binding (84% of the initial bound/free hormone ratio in comparison with normal cells) essentially related to a loss in receptor affinity for insulin. Thus, cultured human keratinocytes which express insulin receptors may be a useful tool in understanding skin pathology related to insulin disorders.

  12. Long-term culture and functional characterization of follicular cells from adult normal human thyroids.

    PubMed Central

    Curcio, F; Ambesi-Impiombato, F S; Perrella, G; Coon, H G

    1994-01-01

    We have obtained long-term cultures of differentiated proliferating follicular cells from normal adult human thyroid glands. In vitro growth of such human cells has been sustained by a modified F-12 medium, supplemented with bovine hypothalamus and pituitary extracts and no added thyrotropin. Cultures have been expanded, cloned, frozen, successfully retrieved, and characterized. Functional characterization of these cells shows constitutive thyroglobulin production and release and thyrotropin-dependent adenosine 3',5'-cyclic monophosphate production, the latter apparently not associated with significant increases in DNA synthesis or cell proliferation. Genetic characterization of these cells by chromosome counting showed the normal diploid chromosome number. The ability to cultivate differentiated human thyroid follicular cells in long-term culture opens possibilities for investigating the transduction pathways of thyrotropin stimulation in normal and pathological human tissues, developing clinically relevant in vitro assays, and considering cellular and molecular therapies. Images PMID:8090760

  13. Regulation of human renin expression in chorion cell primary cultures

    SciTech Connect

    Duncan, K.G.; Haidar, M.A.; Baxter, J.D.; Reudelhuber, T.L. )

    1990-10-01

    The human renin gene is expressed in the kidney, placenta, and several other sites. The release of renin or its precursor, prorenin, can be affected by several regulatory agents. In this study, primary cultures of human placental cells were used to examine the regulation of prorenin release and renin mRNA levels and of the transfected human renin promoter linked to chloramphenicol acetyltransferase reporter sequences. Treatment of the cultures with a calcium ionophore alone, calcium ionophore plus forskolin (that activates adenylate cyclase), or forskolin plus a phorbol ester increased prorenin release and renin mRNA levels 1.3{endash} to 6{endash}fold, but several classes of steroids did not affect prorenin secretion or renin RNA levels. These results suggest that (i) the first 584 base pairs of the renin gene 5'{endash}flanking DNA do not contain functional glucocorticoid or estrogen response elements, (ii) placental prorenin release and renin mRNA are regulated by calcium ion and by the combinations of cAMP with either C kinase or calcium ion, and (iii) the first 100 base pairs of the human renin 5'{endash}flanking DNA direct accurate initiation of transcription and can be regulated by cAMP. Thus, some control of renin release in the placenta (and by inference in other tissues) occurs via transcriptional influences on its promoter.

  14. ATP stimulates calcium influx in primary astrocyte cultures

    SciTech Connect

    Neary, J.T.; van Breemen, C.; Forster, E.; Norenberg, L.O.; Norenberg, M.D.

    1988-12-30

    The effect of ATP and other purines on /sup 45/Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular /sup 45/Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to /sup 45/Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of /sup 45/Ca influx at 90 sec were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl3 blocked the purine-induced /sup 45/Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels.

  15. Political, cultural and economic foundations of primary care in Europe.

    PubMed

    Kringos, Dionne S; Boerma, Wienke G W; van der Zee, Jouke; Groenewegen, Peter P

    2013-12-01

    This article explores various contributing factors to explain differences in the strength of the primary care (PC) structure and services delivery across Europe. Data on the strength of primary care in 31 European countries in 2009/10 were used. The results showed that the national political agenda, economy, prevailing values, and type of healthcare system are all important factors that influence the development of strong PC. Wealthier countries are associated with a weaker PC structure and lower PC accessibility, while Eastern European countries seemed to have used their growth in national income to strengthen the accessibility and continuity of PC. Countries governed by left-wing governments are associated with a stronger PC structure, accessibility and coordination of PC. Countries with a social-security based system are associated with a lower accessibility and continuity of PC; the opposite is true for transitional systems. Cultural values seemed to affect all aspects of PC. It can be concluded that strengthening PC means mobilising multiple leverage points, policy options, and political will in line with prevailing values in a country.

  16. Ultrasonic differentiation of normal versus malignant breast epithelial cells in monolayer cultures

    PubMed Central

    Doyle, Timothy E.; Goodrich, Jeffrey B.; Ambrose, Brady J.; Patel, Hemang; Kwon, Soonjo; Pearson, Lee H.

    2010-01-01

    Normal and malignant mammary epithelial cells were studied using laboratory measurements, wavelet analysis, and numerical simulations of monolayer cell cultures to determine whether microscopic breast cancer can be detected in vitro with high-frequency ultrasound. Pulse-echo waveforms were acquired by immersing a broadband, unfocused 50-MHz transducer in the growth media of cell culture well plates and collecting the first reflection from the well bottoms. The simulations included a multilayer pulse-reflection model and a model of two-dimensional arrays of spherical cells and nuclei. The results show that normal and malignant cells produce time-domain signals and spectral features that are significantly different. PMID:21110531

  17. Elemental composition of normal primary tooth enamel analyzed with XRMA and SIMS.

    PubMed

    Sabel, Nina; Dietz, Wolfram; Lundgren, Ted; Nietzsche, Sandor; Odelius, Hans; Rythén, Marianne; Rizell, Sara; Robertson, Agneta; Norén, Jörgen G; Klingberg, Gunilla

    2009-01-01

    There is an interest to analyze the chemical composition of enamel in teeth from patients with different developmental disorders or syndromes and evaluate possible differences compared to normal composition. For this purpose, it is essential to have reference material. The aim of this study was to, by means of X-ray micro analyses (XRMA) and secondary ion mass spectrometry (SIMS), present concentration gradients for C, O, P and Ca and F, Na, Mg, Cl, K and Sr in normal enamel of primary teeth from healthy individuals. 36 exfoliated primary teeth from 36 healthy children were collected, sectioned, and analyzed in the enamel and dentin with X-ray micro analyses for the content of C, O, P and Ca and F, Na MgCl, K and Sr. This study has supplied reference data for C, O, P and Ca in enamel in primary teeth from healthy subjects. No statistically significant differences in the elemental composition were found between incisors and molars.The ratio Ca/P is in concordance with other studies. Some elements have shown statistically significant differences between different levels of measurement. These results may be used as reference values for research on the chemical composition of enamel and dentin in primary teeth from patients with different conditions and/or syndromes.

  18. Antioxidant enzymes in malignant prostate cell lines and in primary cultured prostatic cells.

    PubMed

    Jung, K; Seidel, B; Rudolph, B; Lein, M; Cronauer, M V; Henke, W; Hampel, G; Schnorr, D; Loening, S A

    1997-01-01

    The antioxidant enzymes catalase, glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were determined in the androgen-response LNCaP and androgen-nonresponsive PC-3 and DU 145 cells as well as in prostatic epithelial cell cultures of benign and malignant human prostatic tissue. There were no differences between the enzyme activities of the human primary cell cultures from cancerous tissue and their normal counterparts. The enzyme activities of the three permanent cell lines were either higher (SOD, catalase, GR) or lower (GST, GPx) than in the primary cell cultures. In LNCaP cells catalase and GR were significantly higher, GST, in contrast, was significantly lower than in PC-3 and DU 145 cells. GST in PC-3 and DU 145 cells, and SOD in all the three cell lines showed no significant differences. Catalase, GPx and GR values were significantly different in the three permanent cell lines. The different enzymatic equipment of the prostate cancer cell lines provides the basis for experimental testing of new concepts of cancer treatment with the help of systematic modulations of the antioxidant defence systems in prostate cancer.

  19. Primary Cultures of Glomerular Parietal Epithelial Cells or Podocytes with Proven Origin

    PubMed Central

    Schulte, Kevin; Sechi, Antonio; Sauer-Lehnen, Sibille; Tag, Carmen; Boor, Peter; Kuppe, Christoph; Warsow, Gregor; Schordan, Sandra; Mostertz, Jörg; Chilukoti, Ravi Kumar; Homuth, Georg; Endlich, Nicole; Tacke, Frank; Weiskirchen, Ralf; Fuellen, Georg; Endlich, Karlhans; Floege, Jürgen; Smeets, Bart; Moeller, Marcus J.

    2012-01-01

    Parietal epithelial cells (PECs) are crucially involved in the pathogenesis of rapidly progressive glomerulonephritis (RPGN) as well as in focal and segmental glomerulosclerosis (FSGS). In this study, transgenic mouse lines were used to isolate pure, genetically tagged primary cultures of PECs or podocytes using FACsorting. By this approach, the morphology of primary glomerular epithelial cells in culture could be resolved: Primary podocytes formed either large cells with intracytoplasmatic extensions or smaller spindle shaped cells, depending on specific culture conditions. Primary PECs were small and exhibited a spindle-shaped or polygonal morphology. In the very early phases of primary culture, rapid changes in gene expression (e.g. of WT-1 and Pax-2) were observed. However, after prolonged culture primary PECs and podocytes still segregated clearly in a transcriptome analysis - demonstrating that the origin of primary cell cultures is important. Of the classical markers, synaptopodin and podoplanin expression were differentially regulated the most in primary PEC and podocyte cultures. However, no expression of any endogenous gene allowed to differentiate between the two cell types in culture. Finally, we show that the transcription factor WT1 is also expressed by PECs. In summary, genetic tagging of PECs and podocytes is a novel and necessary tool to derive pure primary cultures with proven origin. These cultures will be a powerful tool for the emerging field of parietal epithelial cell biology. PMID:22529955

  20. Regulation of heme metabolism in normal and sideroblastic bone marrow cells in culture

    SciTech Connect

    Ibraham, N.G.; Lutton, J.D.; Hoffman, R.; Levere, R.D.

    1985-05-01

    Heme metabolism was examined in developing in vitro erythroid colonies (CFUE) and in bone marrow samples taken directly from four normal donors and four patients with sideroblastic anemia. Maximum activities of delta-aminolevulinic acid synthase (ALAS), ALA dehydratase (ALAD), and /sup 14/C-ALA incorporation into heme were achieved in normal marrow CFUE after 8 days of culture, whereas heme oxygenase progressively decreased to low levels of activity during the same period. Assays on nucleated bone marrow cells taken directly from patients revealed that ALAS activity was considerably reduced in idiopathic sideroblastic anemia (IASA) and X-linked sideroblastic anemia (X-SA) bone marrow specimens, whereas the activity increased more than twofold (normal levels) when cells were assayed from 8-day CFUE. In all cases, ALAD activity appeared to be within normal levels. Measurement of heme synthesis revealed that normal levels of /sup 14/C-ALA incorporation into heme were achieved in IASA cells but were reduced in X-SA cells. In marked contrast to levels in normal cells, heme oxygenase was found to be significantly elevated (two- to fourfold) in bone marrow cells taken directly from patients with IASA and X-SA. Results from this study demonstrate that IASA and X-SA bone marrow cells have disturbances in ALAS and heme metabolism, and that erythropoiesis (CFUE) can be restored to normal levels when cells are cultured in methylcellulose.

  1. Neoplastic cells obtained from Hodgkin's disease are potent stimulators of human primary mixed lymphocyte cultures.

    PubMed

    Fisher, R I; Bostick-Bruton, F; Sauder, D N; Scala, G; Diehl, V

    1983-06-01

    Neoplastic cells obtained from the pleural effusion of a patient with Hodgkin's disease have been maintained in culture since 1978. These tumor cells have been shown to have the cytologic features, cytochemical staining, and cell surface markers of Reed-Sternberg cells. In this study we demonstrate that the cell line termed L428 is a potent stimulator of the primary human mixed lymphocyte reaction. Significant proliferation occurred when mononuclear leukocytes obtained from normal donors were stimulated with radiated L428 cells at responder:stimulator ratios varying from 200:1 to 20:1. Proliferative responses occurred between days 3 and 6 of the cultures with maximal proliferation on day 5. Under optimal culture conditions, mean net proliferative response of 14 normal donors was 51,000 +/- 10,600 dpm. The mixed lymphocyte response was totally blocked by concentrations of monoclonal anti-Ia antibody that had no effect on concanavalin A-induced proliferation. However, the mixed lymphocyte response was not blocked by an anti-K562 cell monoclonal antibody of the same immunoglobulin subclass that binds to the L428 cells. Antigen processing by responder monocytes or Ia-positive cells was not required for the MLC. When responder T cells from two normals were depleted of Ia-bearing cells and monocytes, the mixed lymphocyte reaction between the two normals was eliminated, yet the stimulation of each normal by the L428 cells was not reduced. The cells that proliferated in response to stimulation by the L428 cells were T cells, primarily of the helper subset. No IL 1 activity could be detected in concentrated supernatants of L428 cultures after stimulation of L428 cells by mitogens, phorbol esters, or muramyl dipeptide, or in the MLC. All of these cultures contain fetal calf serum. However, the L428 cells are capable of producing IL 1, because IL 1 was detected when the L428 cells were stimulated with LPS in the absence of fetal calf serum. These neoplastic cells, obtained

  2. Phosphorylation of intracellular proteins related to the multihormonal regulation of prolactin: comparison of normal anterior pituitary cells in culture with the tumor-derived GH cell lines

    SciTech Connect

    Beretta, L.; Boutterin, M.C.; Sobel, A.

    1988-01-01

    We have previously identified a group of cytoplasmic phosphoproteins (proteins 1-11) whose phosphorylation could be related, on a pharmacological basis, to the multihormonal regulation of PRL synthesis and release in the anterior pituitary tumor-derived GH cell lines. Phosphoproteins with identical migration properties on two-dimensional electrophoresis gels were also detectable in normal rat anterior pituitary cells in culture. We designed appropriate culture and (/sup 32/P) phosphate-labeling conditions allowing to analyze the regulation of the phosphorylation of these proteins in normal pituitary cells. TRH, 12-O-tetradecanoylphorbol-13-acetate, and vasoactive intestinal peptide induced the same qualitative changes in phosphorylation of proteins 1-11 in normal as in GH cells. Quantitative differences observed are most likely due to the heterogeneity of primary pituitary cultures. Phosphorylation changes affecting proteins 14-16, not previously detected in GH cells, were also observed with normal anterior pituitary cells. GH cell lines have lost the sensitivity of pituitary lactotrophs for dopamine, an important physiological inhibitor of PRL synthesis and release. In normal anterior pituitary cells in culture, dopamine inhibited also the TRH-stimulated phosphorylation of proteins 1-10, thus strengthening the correlation between phosphorylation of these proteins and multihormonal regulation of pituitary cell functions. Our results indicate: 1) that the same phosphoproteins as in GH cells are related to the multihormonal regulation of nontumoral, normal anterior pituitary cells in culture; 2) that dopamine acts by interfering with the phosphorylation of these proteins.

  3. Accumulation of iron oxide nanoparticles by cultured primary neurons.

    PubMed

    Petters, Charlotte; Dringen, Ralf

    2015-02-01

    Magnetic iron oxide nanoparticles (IONPs) are frequently used for biomedical applications. Although nanoparticles can enter the brain, little is known so far on the uptake of IONPs in neurons and on their neurotoxic potential. Hence, we applied dimercaptosuccinate (DMSA)-coated IONPs to cultured primary rat cerebellar granule neurons. These IONPs had average hydrodynamic diameters of around 80 nm and 120 nm when dispersed in incubation medium in the absence and the presence of 10% fetal calf serum, respectively. Acute exposure of neurons with IONPs for up to 6 h did neither alter the cell morphology nor compromise cell viability, although neurons accumulated large amounts of IONPs in a time- and concentration-dependent manner which caused delayed toxicity. For the first 30 min of incubation of neurons at 37 °C with IONPs the cellular iron content increased proportionally to the concentration of IONPs applied irrespective of the absence and the presence of serum. IONP-exposure in the absence of serum generated maximal cellular iron contents of around 3000 nmol iron/mg protein after 4 h of incubation, while the accumulation in the presence of 10% serum was slower and reached already within 1 h maximal values of around 450 nmol iron/mg protein. For both incubation conditions was the increase in cellular iron contents significantly lowered by reducing the incubation temperature to 4 °C. Application of inhibitors of endocytotic pathways did not affect neuronal IONP accumulation in the absence of serum, while inhibitors of clathrin-mediated endocytosis lowered significantly the IONP accumulation in the presence of serum. These data demonstrate that DMSA-coated IONPs are not acutely toxic to cultured neurons and that a protein corona around the particles strongly affects their interaction with neurons. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Interspecies differences in metabolism of arsenic by cultured primary hepatocytes

    SciTech Connect

    Drobna, Zuzana; Walton, Felecia S.; Harmon, Anne W.; Thomas, David J.; Styblo, Miroslav

    2010-05-15

    Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAs methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [{sup 73}As]arsenite (iAs{sup III}; 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs{sup III} to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs{sup III} than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs{sup III} was associated with inhibition of DMAs production by moderate concentrations of iAs{sup III} and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also contribute to

  5. Spatial distribution of trace element Ca-normalized ratios in primary and permanent human tooth enamel.

    PubMed

    Tacail, Théo; Kovačiková, Lenka; Brůžek, Jaroslav; Balter, Vincent

    2017-12-15

    The trace elements distribution embedded in tooth enamel offers a means to study exposure of toxic metals and allows the reconstruction of dietary behaviors. The quantification of most of the elements with a spatial high resolution (~50μm) is routinely achieved using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). However, the lack of a comprehensive framework of trace elements distribution in enamel jeopardizes any endorsed sampling strategy using LA-ICPMS. The present work is an effort to improve our knowledge on this issue. We studied a suite of 22 sectioned teeth with known dietary history, including 12 3rd molars from 12 living individuals and 10 primary teeth from 3 living individuals. Using LA-ICPMS, we measured Ca, Cu, Zn, Ni, Sr, Ba and Pb variations along 2 or 3 rasters from cervical to occlusal enamel. Calcium concentrations are lower in primary than in permanent teeth and do not vary spatially within a tooth suggesting that enamel matures homogeneously before eruption. The Pb/Ca ratio does not vary within tooth enamel and between primary and permanent tooth enamel. The Cu/Ca and Ni/Ca ratios do not vary within tooth enamel but discriminate primary from permanent tooth enamel. The Zn/Ca ratios are higher in permanent than in primary tooth enamel, and increase up to an order of magnitude in the last hundred of microns at the enamel surface. The Sr/Ca and Ba/Ca ratios are higher in permanent than in primary tooth enamel, and decrease from the enamel-dentine junction towards outer enamel in permanent but not in primary tooth enamel. Considering the Ca-normalized intra-tooth variations of Zn, Sr and Ba, we recommend to perform laser ablation rasters along the enamel-dentine junction because this area is likely to retain most of the original and complete chemical information related to individual's life. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Accumulation of pyrethroid compounds in primary cultures of rat cortical neurons

    EPA Science Inventory

    Recent studies have demonstrated that lipophilic compounds (e.g. methylmercury, polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs)) rapidly accumulate in cells in culture to concentrations much higher than in the surrounding media. Primary cultures of neur...

  7. Accumulation of pyrethroid compounds in primary cultures of rat cortical neurons

    EPA Science Inventory

    Recent studies have demonstrated that lipophilic compounds (e.g. methylmercury, polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs)) rapidly accumulate in cells in culture to concentrations much higher than in the surrounding media. Primary cultures of neur...

  8. Erythropoietin triggers a burst of GATA-1 in normal human erythroid cells differentiating in tissue culture.

    PubMed Central

    Dalyot, N; Fibach, E; Ronchi, A; Rachmilewitz, E A; Ottolenghi, S; Oppenheim, A

    1993-01-01

    GATA-1 is a central transcription-activator of erythroid differentiation. In the present work we have studied the kinetics of its expression and activity during development of normal human erythroid progenitors, grown in primary cultures. In response to the addition of erythropoietin (Epo), the cells undergo proliferation and differentiation in a synchronized fashion. This recently developed experimental system allows biochemical dissection of erythroid differentiation in a physiological meaningful environment. No DNA-binding activity of GATA-1 could be detected before the addition of Epo, although a very low level of mRNA was observed. Following Epo addition there was a sharp parallel rise in both mRNA and DNA-binding activity, consistent with positive autoregulation of the GATA-1 gene. After reaching a peak on day 7-9, both mRNA and protein activity decreased. The binding activity of the ubiquitous factor SP1 showed a biphasic pattern; its second peak usually coincided with the GATA-1 peak, suggesting that SP1 also plays a specific role in erythroid maturation. The highest activity of GATA-1 per erythroid cell was found on day 6-8, immediately preceding the major rise in globin gene mRNA and in the number of hemoglobinized cells. The results imply that a high level of GATA-1 activity is necessary for globin gene expression and erythroid maturation, suggesting that a requirement for a threshold concentration of GATA-1 is part of the mechanism that determines the final steps of erythroid maturation. Images PMID:8371977

  9. Human Airway Primary Epithelial Cells Show Distinct Architectures on Membrane Supports Under Different Culture Conditions.

    PubMed

    Min, Kyoung Ah; Rosania, Gus R; Shin, Meong Cheol

    2016-06-01

    To facilitate drug development for lung delivery, it is highly demanding to establish appropriate airway epithelial cell models as transport barriers to evaluate pharmacokinetic profiles of drug molecules. Besides the cancer-derived cell lines, as the primary cell model, normal human bronchial epithelial (NHBE) cells have been used for drug screenings because of physiological relevance to in vivo. Therefore, to accurately interpret drug transport data in NHBE measured by different laboratories, it is important to know biophysical characteristics of NHBE grown on membranes in different culture conditions. In this study, NHBE was grown on the polyester membrane in a different medium and its transport barrier properties as well as cell architectures were fully characterized by functional assays and confocal imaging throughout the days of cultures. Moreover, NHBE cells on inserts in a different medium were subject to either of air-interfaced culture (AIC) or liquid-covered culture (LCC) condition. Cells in the AIC condition were cultivated on the membrane with medium in the basolateral side only, whereas cells with medium in apical and basolateral sides under the LCC condition. Quantitative microscopic imaging with biophysical examination revealed distinct multilayered architectures of differentiated NHBE cells, suggesting NHBE as functional cell barriers for the lung-targeting drug transport.

  10. Transport Mechanism of Nicotine in Primary Cultured Alveolar Epithelial Cells.

    PubMed

    Takano, Mikihisa; Nagahiro, Machi; Yumoto, Ryoko

    2016-02-01

    Nicotine is absorbed from the lungs into the systemic circulation during cigarette smoking. However, there is little information concerning the transport mechanism of nicotine in alveolar epithelial cells. In this study, we characterized the uptake of nicotine in rat primary cultured type II (TII) and transdifferentiated type I-like (TIL) epithelial cells. In both TIL and TII cells, [(3)H]nicotine uptake was time and temperature-dependent, and showed saturation kinetics. [(3)H]Nicotine uptake in these cells was not affected by Na(+), but was sensitive to extracellular and intracellular pH, suggesting the involvement of a nicotine/proton antiport system. The uptake of [(3)H]nicotine in these cells was potently inhibited by organic cations such as clonidine, diphenhydramine, and pyrilamine, but was not affected by substrates and/or inhibitors of known organic cation transporters such as carnitine, 1-methyl-4-phenylpyridinium, and tetraethylammonium. In addition, the uptake of [(3)H]nicotine in TIL cells was stimulated by preloading the cells with unlabeled nicotine, pyrilamine, and diphenhydramine, but not with tetraethylammonium. These results suggest that a novel proton-coupled antiporter is involved in the uptake of nicotine in alveolar epithelial cells and its absorption from the lungs into the systemic circulation.

  11. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes.

    PubMed

    Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

    2016-11-30

    The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

  12. Mitochondrial-dependent manganese neurotoxicity in rat primary astrocyte cultures

    PubMed Central

    Yin, Zhaoobao; Aschner, Judy L.; Santos, Ana Paula dos; Aschner, Michael

    2008-01-01

    Chronic exposure to excessive levels of Mn results in a movement disorder termed manganism, which resembles Parkinson’s disease (PD). The pathogenic mechanisms underlying this disorder are not fully understood. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity. In the present study, we investigated the effects of Mn on mitochondrial function. Primary astrocyte cultures were prepared from cerebral cortices of one-day-old Sprague–Dawley rats. We have examined the cellular toxicity of Mn and its effects on the phosphorylation of extracellular signal-regulated kinase (ERK) and activation of the precursor protein of caspase-3. The potentiometric dye, tetramethylrhodamine ethyl ester (TMRE), was used to assess the effect of Mn on astrocytic mitochondrial inner membrane potential (ΔΨm). Our studies show that, in a concentration-dependent manner, Mn induces significant (p<0.05) activation of astrocyte caspase-3 and phosphorylated extracellular signal-regulated kinase (p-ERK). Mn treatment (1 and 6 hrs) also significantly (p<0.01) dissipates the ΔΨm in astrocytes as evidenced by a decrease in mitochondrial TMRE fluorescence. These results suggest that activations of astrocytic caspase-3 and ERK are involved in Mn-induced neurotoxicity via mitochondrial-dependent pathways. PMID:18313649

  13. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

    PubMed Central

    Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

    2016-01-01

    The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes. PMID:27916916

  14. Measurement of Primary Ejecta From Normal Incident Hypervelocity Impact on Lunar Regolith Simulant

    NASA Technical Reports Server (NTRS)

    Edwards, David L.; Cooke, William; Moser, Danielle; Swift, Wesley

    2007-01-01

    The National Aeronautics and Space Administration (NASA) continues to make progress toward long-term lunar habitation. Critical to the design of a lunar habitat is an understanding of the lunar surface environment. A subject for further definition is the lunar primary ejecta environment. The document NASA SP-8013 was developed for the Apollo program and is the latest definition of the primary ejecta environment. There is concern that NASA SP-8013 may over-estimate the lunar primary ejecta environment. NASA's Meteoroid Environment Office (MEO) has initiated several tasks to improve the accuracy of our understanding of the lunar surface primary ejecta environment. This paper reports the results of experiments on projectile impact into pumice targets, simulating lunar regolith. The Ames Vertical Gun Range (AVGR) was used to accelerate spherical Pyrex projectiles of 0.29g to velocities ranging between 2.5 km/s and 5.18 km/s. Impact on the pumice target occurred at normal incidence. The ejected particles were detected by thin aluminum foil targets placed around the pumice target in a 0.5 Torr vacuum. A simplistic technique to characterize the ejected particles was formulated. Improvements to this technique will be discussed for implementation in future tests.

  15. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    SciTech Connect

    Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

    2011-08-26

    Highlights: {yields} Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. {yields} The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. {yields} Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  16. Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

    PubMed

    Lopachev, A V; Lopacheva, O M; Abaimov, D A; Koroleva, O V; Vladychenskaya, E A; Erukhimovich, A A; Fedorova, T N

    2016-05-01

    Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.

  17. Effect of lectin from Chelidonium majus L. on normal and cancer cells in culture.

    PubMed

    Fik, E; Wołuń-Cholewa, M; Kistowska, M; Warchoł, J B; Goździcka-Józefiak, A

    2001-01-01

    Lectin from Chelidonium majus L. (CML) significantly stimulates the proliferation of human lymphocytes and has hemagglutination activity towards group B human erythrocytes and potent antimicrobial properties against multiresistant enterococci and staphylococci. In the present work we describe the effect of lectin from Chelidonium majus L on normal and cancercells in culture in vitro. The studies were performed on three types of cells: CHO, R2C and on normal mouse fibroblasts. Effects on the cultures were examined 24 h after addition of CML. Exposure to CML resulted in growth inhibition of CHO and R2C cells but not of fibroblasts. Moreover, evident apoptotic lesions were observed in CHO cells and less well marked apoptotic lesions in R2C cells. In contrast, only insignificant numbers of fibroblasts reacted to the applied lectin.

  18. A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro.

    PubMed

    Schlaermann, Philipp; Toelle, Benjamin; Berger, Hilmar; Schmidt, Sven C; Glanemann, Matthias; Ordemann, Jürgen; Bartfeld, Sina; Mollenkopf, Hans J; Meyer, Thomas F

    2016-02-01

    Helicobacter pylori is the causative agent of gastric diseases and the main risk factor in the development of gastric adenocarcinoma. In vitro studies with this bacterial pathogen largely rely on the use of transformed cell lines as infection model. However, this approach is intrinsically artificial and especially inappropriate when it comes to investigating the mechanisms of cancerogenesis. Moreover, common cell lines are often defective in crucial signalling pathways relevant to infection and cancer. A long-lived primary cell system would be preferable in order to better approximate the human in vivo situation. Gastric glands were isolated from healthy human stomach tissue and grown in Matrigel containing media supplemented with various growth factors, developmental regulators and apoptosis inhibitors to generate long-lasting normal epithelial cell cultures. Culture conditions were developed which support the formation and quasi-indefinite growth of three dimensional (3D) spheroids derived from various sites of the human stomach. Spheroids could be differentiated to gastric organoids after withdrawal of Wnt3A and R-spondin1 from the medium. The 3D cultures exhibit typical morphological features of human stomach tissue. Transfer of sheared spheroids into 2D culture led to the formation of dense planar cultures of polarised epithelial cells serving as a suitable in vitro model of H. pylori infection. A robust and quasi-immortal 3D organoid model has been established, which is considered instrumental for future research aimed to understand the underlying mechanisms of infection, mucosal immunity and cancer of the human stomach. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  19. L-carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts.

    PubMed

    Shao, Lan; Li, Qing-Huan; Tan, Zheng

    2004-11-12

    Telomere is the repetitive DNA sequence at the end of chromosomes, which shortens progressively with cell division and limits the replicative potential of normal human somatic cells. L-carnosine, a naturally occurring dipeptide, has been reported to delay the replicative senescence, and extend the lifespan of cultured human diploid fibroblasts. In this work, we studied the effect of carnosine on the telomeric DNA of cultured human fetal lung fibroblast cells. Cells continuously grown in 20 mM carnosine exhibited a slower telomere shortening rate and extended lifespan in population doublings. When kept in a long-term nonproliferating state, they accumulated much less damages in the telomeric DNA when cultured in the presence of carnosine. We suggest that the reduction in telomere shortening rate and damages in telomeric DNA made an important contribution to the life-extension effect of carnosine.

  20. Dual effects of carnosine on energy metabolism of cultured cortical astrocytes under normal and ischemic conditions.

    PubMed

    Shen, Yao; Tian, Yueyang; Yang, Jianbo; Shi, Xiaojie; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

    2014-01-01

    The aim of this study was to investigate the effects of carnosine on the bioenergetic profile of cultured cortical astrocytes under normal and ischemic conditions. The Seahorse Bioscience XF96 Extracellular Flux Analyzer was used to measure the oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs) of cultured cortical astrocytes treated with and without carnosine under normal and ischemic conditions. Under the normal growth condition, the basal OCRs and ECARs of astrocytes were 21.72±1.59 pmol/min/μg protein and 3.95±0.28 mpH/min/μg protein respectively. Mitochondrial respiration accounted for ~80% of the total cellular respiration and 85% of this coupled to ATP synthesis. Carnosine significantly reduced basal OCRs and ECARs and ATP-linked respiration, but it strikingly increased the spare respiratory capacity of astrocytes. The cellular ATP level in carnosine-treated astrocytes was reduced to ~42% of the control. However, under the ischemic condition, carnosine upregulated the mitochondrial respiratory and cellular ATP content of astrocytes exposed to 8h of oxygen-glucose deprivation (OGD) followed by 24 h of recovery under the normal growth condition. Carnosine may be an endogenous regulator of astrocyte energy metabolism and a clinically safe therapeutic agent for promoting brain energy metabolism recovery after ischemia/reperfusion injury. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Effect of Organizational Culture on Patient Access, Care Continuity, and Experience of Primary Care.

    PubMed

    Hung, Dorothy; Chung, Sukyung; Martinez, Meghan; Tai-Seale, Ming

    2016-01-01

    This study examined relationships between organizational culture and patient-centered outcomes in primary care. Generalized least squares regression was used to analyze patient access, care continuity, and reported experiences of care among 357 physicians in 41 primary care departments. Compared with a "Group-oriented" culture, a "Rational" culture type was associated with longer appointment wait times, and both "Hierarchical" and "Developmental" culture types were associated with less care continuity, but better patient experiences with care. Understanding the unique effects of organizational culture can enhance the delivery of more patient-centered care.

  2. Exploring Culture in Locally Published English Textbooks for Primary Education in Turkey

    ERIC Educational Resources Information Center

    Kirkgöz, Yasemin; Agçam, Reyhan

    2011-01-01

    Since language and culture are closely interwoven, the integration of culture into textbooks used for teaching English as a second/foreign language has become a widely accepted phenomenon. This study investigates the cultural elements in locally published English textbooks used for Turkish primary schools following two major curriculum innovations…

  3. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells

    PubMed Central

    1980-01-01

    We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates. PMID:7372714

  4. Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts.

    PubMed Central

    Kuhnlein, U; Lee, B; Linn, S

    1978-01-01

    Uracil DNA N-glycosidase, an enzyme which participates in the excision of uracil from DNA, was measured in extracts from fibroblasts lines cultured from normal subjects, from several subjects with the genetic disease xeroderma pigmentosum, and from a subject with ataxia telangiectasia. The cell lines representative of complementation groups A and D of xeroderma pigmentosum and of ataxia telangiectasia had roughly the same level of activity as did the normal cells. On the other hand, cells from two xeroderma pigmentosum variants (XP4BE and XP13BE) had roughly half the normal level of activity, and cells from the heterozygous mother of XP4BE had an intermediate level of activity. In spite of these quantitative differences, no systematic alterations in reaction characteristics, apparent Km for substrate, or purification characteristics were noted for enzyme from any of the lines. Thus a causal relationship, if any, between levels of activity and the disease symptoms is equivocal. PMID:643602

  5. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells.

    PubMed

    Virtanen, I; Ekblom, P; Laurila, P

    1980-05-01

    We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates.

  6. [A comparison study of pulsitile ocular blood flow in normal eyes and primary open angle glaucoma].

    PubMed

    Zhang, Ming-Zhi; Fu, Zhi-Fu; Liu, Xiao-Rui; Zheng, C

    2004-04-01

    To compare pulsatile ocular blood flow (POBF) and intraocular pressure (IOP) in primary open angle glaucoma (POAG) and normal control group matched for age, sex and refraction error, to investigate the rule of hem-dynamics in POAG and to determine the sensitivity and specificity of POBF measurement as a diagnostic test for glaucoma. Prior to the test a questionnaire was completed to determine age, sex, refractive error, family history of glaucoma, history of eye diseases, ocular medication, medical history and using of systemic beta-blockers. Patients of POAG were determined by following diagnostics standards: (1) Three IOP >25 mm Hg in different times of one day. (2) The fluctuate of IOP > 8 mm Hg during 24 hours. (3) Typical glaucoma changes in the visual field. (4) Typical glaucoma changes in optic disc. There were 100 POAG subjects with single eye observed (50 male and 50 female). We picked up 100 eyes randomly (50 male and 50 female) in 534 normal persons who matched for following conditions: (1) Sex. (2) Discrepancy of age less than 5 years. (3) Discrepancy of the refraction error less than +/- 2.00 DS. as the normal comparison group. The tonometer used was the POBF Tonometry. Pulse amplitude of IOP (PA), pulsatile ocular blood flow (POBF), pulse/heart rate (PR), maximum-IOP (max-IOP), minimum-IOP (min-IOP) and average IOP (aver-IOP) were obtained before the medical therapy and 1 or 2 weeks after the operation. The correlation between the POBF & mean value of the perimeter was analyzed. POBF was analyzed to determine the sensitivity and specificity of POBF measurement as a diagnostic test. The value of POBF in POAG and normal control was (9.72 +/- 3.47) microl/s and (12.04 +/- 4.68) microl/s, respectively. POAG patients' POBF, PV, PA, and AVE-IOP were less than those in the normal control, and the difference was statistically significant. There was no statistically significant correlation between the changes of visual field and POBF (r = 0.224, P = 0

  7. Proton NMR characterization of intact primary and metastatic melanoma cells in 2D & 3D cultures.

    PubMed

    Ramachandran, Gokula Krishnan; Yeow, Chen Hua

    2017-03-16

    To characterize the differences between the primary and metastatic melanoma cell lines grown in 2D cultures and 3D cultures. Primary melanoma cells (WM115) and metastatic melanoma cells (WM266) extracted from a single donor was cultured in 2D as well as 3D cultures. These cells were characterized using proton NMR spectrometry, and the qualitative chemical shifts markers were identified and discussed. In monolayer culture (2D), we observed one qualitative chemical shift marker for primary melanoma cells. In spheroid cultures (3D), we observed nine significant chemical shifts, of which eight markers were specific for primary melanoma spheroids, whereas the other one marker was specific to metastatic melanoma spheroids. This study suggests that the glucose accumulation and phospholipid composition vary significantly between the primary and metastatic cells lines that are obtained from a single donor and also with the cell culturing methods. 14 qualitative chemical shift markers were obtained in the comparison between monolayer culture and spheroids cultures irrespective of the differences in the cell lines. Among which 4 were unique to monolayer cultures whereas 10 chemical shifts were unique to the spheroid cultures. This study also shows that the method of cell culture would drastically affect the phospholipid composition of the cells and also depicts that the cells in spheroid culture closely resembles the cells in vivo. This study shows the high specificity of proton NMR spectrometry in characterizing cancer cell lines and also shows the variations in the glucose accumulation and phospholipid composition between the primary and metastatic melanoma cell lines from the same donor. Differences in the cell culture method does plays an important role in phospholipid composition of the cells.

  8. Effects of nutritional and hormonal factors on the metabolism of retinol-binding protein by primary cultures of rat hepatocytes

    SciTech Connect

    Dixon, J.L.; Goodman, D.S.

    1987-01-01

    Studies were conducted to explore hormonal and nutritional factors that might be involved in the regulation of retinol-binding protein (RBP) synthesis and secretion by the liver. The studies employed primary cultures of hepatocytes from normal rats. When cells were cultured in Dulbecco's modified Eagle's medium alone, a high rate of RBP secretion was observed initially, which declined and became quite low by 24 hr. Supplementing the medium with amino acids maintained RBP and albumin secretion at moderate (but less than initial) rates for at least 3 days. Further addition of dexamethasone maintained the production and secretion rates of RBP, transthyretin, and albumin close to the initial rates for up to 3-5 days in culture as measured by radioimmunoassay. Hormonally treated hepatocytes produced and secreted RBP, transthyretin, and albumin at both absolute and relative rates similar to physiological values, as estimated from rates reported by others from studies in vivo and with perfused livers. Glucagon addition partially maintained the secretion rates of these 3 proteins, but less effectively than did dexamethasone. A number of other hormones, added singly or in combination, did not affect RBP production or secretion. Addition of retinol to the cultured normal hepatocytes was without effect upon RBP secretion. These studies show that supplementing the culture medium of hepatocytes with amino acids and dexamethasone maintains RBP production and secretion for several days. In normal hepatocytes, with ample supply of retinol available within the cell, addition of exogenous retinol does not appear to influence RBP metabolism or secretion by the cells.

  9. Effect of betaxolol on primary open-angle glaucoma and normal-tension glaucoma patients.

    PubMed

    Saccà, S C; Macrì, A; Rolando, M; Ciurlo, G

    1998-06-01

    This study is aimed at establishing the efficacy of the therapeutic agent, betaxolol, in diurnal control of IOP (intraocular pressure). Therapy was performed on 32 eyes affected by POAG (primary open-angle glaucoma) and 16 eyes affected by NTG (normal-tension glaucoma). Two preparations of betaxolol were utilized: betaxolol hydrochloride 0.50% (Betoptic) was administered to 15 POAG and 7 NTG eyes; betaxolol hydrochloride 0.25% ophthalmic suspension (Betoptic S) was administered to 17 POAG and 9 NTG eyes. IOP measurements were taken every two hours from 8 a.m. to 8 p.m. IOP was measured before therapy and at 12 hours, 30 days, and 3 months of therapy. Betaxolol hydrochloride 0.50% was more effective at lowering IOP during the day. Diurnal pressure peaks, which are a risk factor concerning the maintenance of visual field in glaucoma patients, were also reduced using 0.50% betaxolol hydrochloride.

  10. Primary ciliary dyskinesia: critical evaluation of clinical symptoms and diagnosis in patients with normal and abnormal ultrastructure

    PubMed Central

    2014-01-01

    Background Primary ciliary dyskinesia (PCD) is a rare disorder with variable disease progression. To date, mutations in more than 20 different genes have been found. At present, PCD subtypes are described according to the ultrastructural defect on transmission electron microscopy (TEM) of the motile cilia. PCD with normal ultrastructure (NU) is rarely reported because it requires additional testing. Biallelic mutations in DNAH11 have been described as one cause of PCD with NU. The aim of our study was to describe the clinical characteristics of a large population of patients with PCD, in relation to the ultrastructural defect. Additionally, we aimed to demonstrate the need for biopsy and cell culture to reliably diagnose PCD, especially the NU subtype. Methods We retrospectively analyzed data from 206 patients with PCD. We compared the clinical characteristics, lung function, microbiology and imaging results of 68 patients with PCD and NU to those of 90 patients with dynein deficiencies and 41 patients with central pair abnormalities. In addition, we aimed to demonstrate the robustness of the diagnosis of the NU subtype in cell culture by data from genetic analysis. Results PCD with NU comprised 33% (68/206) of all patients with PCD. Compared to other subtypes, patients with PCD and NU had a similar frequency of upper and lower respiratory tract problems, as well as similar lung function and imaging. With the currently widely applied approach, without cell culture, the diagnosis would have been missed in 16% (11/68) of patients with NU. Genetic analysis was performed in 29/68 patients with PCD and NU, and biallelic mutations were found in 79% (23/29) of tested patients. Conclusions We reported on the clinical characteristics of a large population of patients with PCD and NU. We have shown that systematic performance of biopsy and cell culture increases sensitivity to detect PCD, especially the subtype with NU. PCD with NU has similar clinical characteristics as

  11. Establishment of a primary culture method for mouse intestinal epithelial cells by organ culture of fetal small intestine.

    PubMed

    Yamada, Kiyoshi; Sato, Kanako; Morishita, Satoru; Kaminogawa, Shuichi; Totsuka, Mamoru

    2009-08-01

    Studies of the physiological functions of intestinal epithelial cells (IECs) have been limited by the difficulty of primary culture of IEC. We established a method for primary culture of mouse IEC by culturing fragments of fetal small intestines pretreated with EDTA. This method reproducibly resulted in the expansion of cytokeratin-positive epithelial cells, and vigorous expansion of the epithelial cells was observed only from intestinal fragments of embryonic days 15-16. These cells expressed alkaline phosphatase activity and major histocompatibility complex (MHC) class II molecules, indicating the mature phenotype of IEC in a small intestine. The cells also presented antigens to CD4(+) T cells. Furthermore, the cells expressed various cytokines and chemokines, and the expression was enhanced by bacterial stimulation. These results indicate that the primary-cultured mouse IEC prepared by the method established here can be a beneficial tool in study of the functions of IECs, especially in mucosal immunity.

  12. Calcium currents and transients in co-cultured contracting normal and Duchenne muscular dystrophy human myotubes.

    PubMed

    Imbert, N; Vandebrouck, C; Duport, G; Raymond, G; Hassoni, A A; Constantin, B; Cullen, M J; Cognard, C

    2001-07-15

    1. The goal of the present study was to investigate differences in calcium movements between normal and Duchenne muscular dystrophy (DMD) human contracting myotubes co-cultured with explants of rat spinal cord with attached dorsal root ganglia. Membrane potential, variations of intracellular calcium concentration and T- and L-type calcium currents were recorded. Further, a descriptive and quantitative study by electron microscopy of the ultrastructure of the co-cultures was carried out. 2. The resting membrane potential was slightly less negative in DMD (-61.4 +/- 1.1 mV) than in normal myotubes (-65.5 +/- 0.9 mV). Both types of myotube displayed spontaneous action potentials (mean firing frequency, 0.42 and 0.16 Hz, respectively), which triggered spontaneous calcium transients measured with Indo-1. 3. The time integral under the spontaneous Ca(2+) transients was significantly greater in DMD myotubes (97 +/- 8 nM s) than in normal myotubes (67 +/- 13 nM s). 4. The L- and T-type current densities estimated from patch-clamp recordings were smaller in DMD cells (2.0 +/- 0.5 and 0.90 +/- 0.19 pA pF(-1), respectively) than in normal cells (3.9 +/- 0.7 and 1.39 +/- 0.30 pA pF(-1), respectively). 5. The voltage-dependent inactivation relationships revealed a shift in the conditioning potential at which inactivation is half-maximal (V(h,0.5)) of the T- and L-type currents towards less negative potentials, from -72.1 +/- 0.7 and -53.7 +/- 1.5 mV in normal cells to -61.9 +/- 1.4 and -29.2 +/- 1.4 mV in DMD cells, respectively. 6. Both descriptive and quantitative studies by electron microscopy suggested a more advanced development of DMD myotubes as compared to normal ones. This conclusion was supported by the significantly larger capacitance of the DMD myotubes (408 +/- 45 pF) than of the normal myotubes (299 +/- 34 pF) of the same apparent size. 7. Taken together, these results show that differences in T- and L-type calcium currents between normal and DMD myotubes cannot

  13. Calcium currents and transients in co-cultured contracting normal and Duchenne muscular dystrophy human myotubes

    PubMed Central

    Imbert, Nathalie; Vandebrouck, Clarisse; Duport, Gérard; Raymond, Guy; Hassoni, Abdul A; Constantin, Bruno; Cullen, Michael J; Cognard, Christian

    2001-01-01

    The goal of the present study was to investigate differences in calcium movements between normal and Duchenne muscular dystrophy (DMD) human contracting myotubes co-cultured with explants of rat spinal cord with attached dorsal root ganglia. Membrane potential, variations of intracellular calcium concentration and T- and L-type calcium currents were recorded. Further, a descriptive and quantitative study by electron microscopy of the ultrastructure of the co-cultures was carried out. The resting membrane potential was slightly less negative in DMD (−61.4 ± 1.1 mV) than in normal myotubes (−65.5 ± 0.9 mV). Both types of myotube displayed spontaneous action potentials (mean firing frequency, 0.42 and 0.16 Hz, respectively), which triggered spontaneous calcium transients measured with Indo-1. The time integral under the spontaneous Ca2+ transients was significantly greater in DMD myotubes (97 ± 8 nm s) than in normal myotubes (67 ± 13 nm s). The L- and T-type current densities estimated from patch-clamp recordings were smaller in DMD cells (2.0 ± 0.5 and 0.90 ± 0.19 pA pF−1, respectively) than in normal cells (3.9 ± 0.7 and 1.39 ± 0.30 pA pF−1, respectively). The voltage-dependent inactivation relationships revealed a shift in the conditioning potential at which inactivation is half-maximal (Vh,0.5) of the T- and L-type currents towards less negative potentials, from −72.1 ± 0.7 and −53.7 ± 1.5 mV in normal cells to −61.9 ± 1.4 and −29.2 ± 1.4 mV in DMD cells, respectively. Both descriptive and quantitative studies by electron microscopy suggested a more advanced development of DMD myotubes as compared to normal ones. This conclusion was supported by the significantly larger capacitance of the DMD myotubes (408 ± 45 pF) than of the normal myotubes (299 ± 34 pF) of the same apparent size. Taken together, these results show that differences in T- and L-type calcium currents between normal and DMD myotubes cannot simply explain all observed

  14. Production of Normal Mammalian Organ Culture Using a Medium Containing Mem-Alpha, Leibovitz L 15, Glucose Galactose Fructose

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor); Prewett, Tacey L. (Inventor)

    1999-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under micro- gravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel. The medium used for culturing the cells, especially a mixture of epithelial and mesenchymal cells contains a mixture of Mem-alpha and Leibovits L15 supplemented with glucose, galactose and fructose.

  15. Cultural democracy: the way forward for primary care of hard to reach New Zealanders.

    PubMed

    Finau, Sitaleki A; Finau, Eseta

    2007-09-01

    The use of cultural democracy, the freedom to practice one's culture without fear, as a framework for primary care service provision is essential for improved health service in a multi cultural society like New Zealand. It is an effective approach to attaining health equity for all. Many successful health ventures are ethnic specific and have gone past cultural competency to the practice of cultural democracy. That is, the services are freely taking on the realities of clients without and malice from those of other ethnicities. In New Zealand the scientific health service to improve the health of a multi cultural society are available but there is a need to improve access and utilization by hard to reach New Zealanders. This paper discusses cultural democracy and provide example of how successful health ventures that had embraced cultural democracy were implemented. It suggests that cultural democracy will provide the intellectual impetus and robust philosophy for moving from equality to equity in health service access and utilization. This paper would provide a way forward to improved primary care utilization, efficiency, effectiveness and equitable access especially for the hard to reach populations. use the realities of Pacificans in New Zealand illustrate the use of cultural democracy, and thus equity to address the "inverse care law" of New Zealand. The desire is for primary care providers to take cognizance and use cultural democracy and equity as the basis for the design and practice of primary health care for the hard to reach New Zealanders.

  16. Stored erythrocytes have less capacity than normal erythrocytes to support primary haemostasis.

    PubMed

    Reinhart, Walter H; Zehnder, Linda; Schulzki, Thomas

    2009-04-01

    Primary haemostasis is mediated by platelet aggregation. Red blood cells (RBCs) are involved in this process. We hypothesised that stored RBCs could have less capacity to support primary haemostasis. This was tested with RBC units from 17 healthy volunteers stored for 45 days. Fresh citrated blood was taken again from the same donors and platelet-rich plasma was prepared, in which RBCs were resuspended with a constant haematocrit (40%), but changing fractions of stored versus fresh autologous RBCs (0, 25, 50, 75, and 100%, respectively). A platelet function analyser PFA-100((R)) was used. In this instrument blood is aspirated through a membrane pore coated with collagen and either epinephrine (EPI) or ADP, which causes platelets to adhere, aggregate, and form an occluding plug, which stops blood flow and is measured as closure time (CT). We found that the CT increased with increasing fractions of stored blood. CT-EPI was 121 +/- 17 seconds [s], 129 +/- 32 s, 164 +/- 45 s (p < 0.000, ANOVA), 214 +/- 54 s (p < 0.0001), and 273 +/- 36 s (p < 0.0001) for 0, 25, 50, 75, and 100% stored RBCs. For CT-ADP the values were 91 +/- 22 s, 95 +/- 12 s, 101 +/- 13 s, 124 +/- 44 s (p = 0.004), and 191 +/- 72 s (p < 0.0001), respectively. We conclude that stored RBCs have less capacity than normal RBCs to support primary haemostasis by platelet aggregation in vitro, suggesting a decreased capacity of stored RBCs to bring platelets into close contact with the wall, which may contribute to sustained bleeding seen after mass transfusion.

  17. The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture

    PubMed Central

    1983-01-01

    Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin. PMID:6404908

  18. Apical vacuole formation by gastric parietal cells in primary culture: effect of low extracellular Ca2+

    PubMed Central

    Nakada, Stephanie L.; Machen, Terry E.; Forte, John G.

    2012-01-01

    In primary culture, the gastric parietal cell's deeply invaginated apical membrane, seen in microscopy by phalloidin binding to F-actin (concentrated in microvilli and a subapical web), is engulfed into the cell, separated from the basolateral membrane (which then becomes the complete plasma membrane), and converted, from a lacy interconnected system of canaliculi, into several separate vacuoles. In this study, vacuolar morphology was achieved by 71% of parietal cells 8 h after typical collagenase digestion of rabbit gastric mucosa, but the tight-junctional protein zonula occludens-1 (ZO-1) was completely delocalized after ∼2 h, when cells were ready for culturing. Use of low-Ca2+ medium (4 mM EGTA) to release cells quickly from gastric glands yielded parietal cells in which ZO-1 was seen in a small spot or ring, a localization quickly lost if these cells were then cultured in normal Ca2+ but remaining up to 20 h if they were cultured in low Ca2+. The cells in low Ca2+ mostly retained, at 20 h, an intermediate morphology of many bulbous canalicular expansions (“prevacuoles”), seemingly with narrow interconnections. Histamine stimulation of 20-h cells with intermediate morphology caused colocalization of proton-pumping H-K-ATPase with canaliculi and prevacuoles but little swelling of those structures, consistent with a remaining apical pore through which secreted acid could escape. Apparent canalicular interconnections, lack of stimulated swelling, and lingering ZO-1 staining indicate inhibition of membrane fission processes that separate apical from basolateral membrane and vacuoles from each other, suggesting an important role for extracellular Ca2+ in these, and possibly other, endocytotic processes. PMID:23099641

  19. Pericellular oxygen concentration of cultured primary human trophoblasts

    PubMed Central

    Chen, Baosheng; Longtine, Mark S.; Nelson, D. Michael

    2012-01-01

    Introduction Oxygen is pivotal in placental development and function. In vitro culture of human trophoblasts provides a useful model to study this phenomenon, but a hotly debated issue is whether or not the oxygen tension of the culture conditions mimics in vivo conditions. We tested the hypothesis that ambient oxygen tensions in culture reflect the pericellular oxygen levels. Methods We used a microelectrode oxygen sensor to measure the concentration of dissolved oxygen in the culture medium equilibrated with 21%, 8% or <0.5% oxygen. Results The concentration of oxygen in medium without cells resembled that in the ambient atmosphere. The oxygen concentration present in medium bathing trophoblasts was remarkably dependent on the depth within the medium where sampling occurred, and the oxygen concentration within the overlying atmosphere was not reflected in medium immediately adjacent to the cells. Indeed, the pericellular oxygen concentration was in a range that most would consider severe hypoxia, at ≤ 0.6% oxygen or about 4.6 mm Hg, when the overlying atmosphere was 21% oxygen. Conclusions We conclude that culture conditions of 21% oxygen are unable to replicate the pO2 of 40–60 mm Hg commonly attributed to the maternal blood in the intervillous space in the second and third trimesters of pregnancy. We further surmise that oxygen atmospheres in culture conditions between 0.5% and 21% provide different oxygen fluxes in the immediate pericellular environment yet can still yield insights into the responses of human trophoblast to different oxygen conditions. PMID:23211472

  20. A meta-ethnography of organisational culture in primary care medical practice.

    PubMed

    Grant, Suzanne; Guthrie, Bruce; Entwistle, Vikki; Williams, Brian

    2014-01-01

    Over the past decade, there has been growing international interest in shaping local organisational cultures in primary healthcare. However, the contextual relevance of extant culture assessment instruments to the primary care context has been questioned. The aim of this paper is to derive a new contextually appropriate understanding of the key dimensions of primary care medical practice organisational culture and their inter-relationship through a synthesis of published qualitative research. A systematic search of six electronic databases followed by a synthesis using techniques of meta-ethnography involving translation and re-interpretation. A total of 16 papers were included in the meta-ethnography from the UK, the USA, Canada, Australia and New Zealand that fell into two related groups: those focused on practice organisational characteristics and narratives of practice individuality; and those focused on sub-practice variation across professional, managerial and administrative lines. It was found that primary care organisational culture was characterised by four key dimensions, i.e. responsiveness, team hierarchy, care philosophy and communication. These dimensions are multi-level and inter-professional in nature, spanning both practice and sub-practice levels. The research contributes to organisational culture theory development. The four new cultural dimensions provide a synthesized conceptual framework for researchers to evaluate and understand primary care cultural and sub-cultural levels. The synthesised cultural dimensions present a framework for practitioners to understand and change organisational culture in primary care teams. The research uses an innovative research methodology to synthesise the existing qualitative research and is one of the first to develop systematically a qualitative conceptual framing of primary care organisational culture.

  1. The selective effect of plasma activated medium in an in vitro co-culture of liver cancer and normal cells

    NASA Astrophysics Data System (ADS)

    Duan, J.; Lu, X.; He, G.

    2017-01-01

    In this work, a co-culture system with liver cancer cell line HepG2 and normal cell line L02 is used to investigate the selective effect on cancer and normal cells by plasma activated medium (PAM), which is closer to the real environment where cancer cells develop. Besides, the co-culture system is a better model to study the selective effect than the widely used separate culture systems, where the cancer cell line and normal cell line are cultured independently. By using the co-culture system, it is found that there is an optimum dose of PAM to induce significant cancer cell apoptosis while keeping minimum damage to normal cells.

  2. Influence of palm oil on doxorubicin induced cytotoxicity in normal and tumor cell cultures.

    PubMed

    Postescu, Ion Dan; Virag, Piroska; Achim, Marcela; Fischer-Fodor, Eva

    2010-01-01

    The vitamin E of palm oil, unlike most other vegetal fats, consists largely of tocotrienols (TT), products previously reported as having antioxidant and tumor-inhibitory properties. A tocotrienols containing palm oil, in the form of liposomes entrapping dosages of 0.5-0.05 microgTT/mL, was studied in combined treatments with doxorubicin (30 min before drug administration). The IC(50) values of doxorubicin, at 24 h, showed that its cytotoxic effects were decreased by palm oil, in a dose effect relationship (p < 0.01, ANOVA), in both normal (Hfl-1, Huvec) and tumor (HepG2, Mls) cells. These results demonstrated an unselective protective activity of tocotrienols, in vitro, on some normal and tumor cultured cells treated with doxorubicin. (c) 2009 John Wiley & Sons, Ltd.

  3. An Air-Liquid Interface Culture System for 3D Organoid Culture of Diverse Primary Gastrointestinal Tissues.

    PubMed

    Li, Xingnan; Ootani, Akifumi; Kuo, Calvin

    2016-01-01

    Conventional in vitro analysis of gastrointestinal epithelium usually relies on two-dimensional (2D) culture of epithelial cell lines as monolayer on impermeable surfaces. However, the lack of context of differentiation and tissue architecture in 2D culture can hinder the faithful recapitulation of the phenotypic and morphological characteristics of native epithelium. Here, we describe a robust long-term three-dimensional (3D) culture methodology for gastrointestinal culture, which incorporates both epithelial and mesenchymal/stromal components into a collagen-based air-liquid interface 3D culture system. This system allows vigorously expansion of primary gastrointestinal epithelium for over 60 days as organoids with both proliferation and multilineage differentiation, indicating successful long-term intestinal culture within a microenvironment accurately recapitulating the stem cell niche.

  4. Transformation of adult rat cardiac myocytes in primary culture.

    PubMed

    Banyasz, Tamas; Lozinskiy, Ilya; Payne, Charles E; Edelmann, Stephanie; Norton, Byron; Chen, Biyi; Chen-Izu, Ye; Izu, Leighton T; Balke, C William

    2008-03-01

    We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long-term cell culture. Morphometric parameters, sarcomere length, T-tubule density, cell capacitance, L-type calcium current (I(Ca,L)), inward rectifier potassium current (I(K1)), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 +/- 70 microm(2) on day 0 to 1432 +/- 50 microm(2) on day 5), T-tubule density, systolic shortening (from 0.11 +/- 0.02 to 0.05 +/- 0.01 microm) and amplitude of calcium transients (from 1.54 +/- 0.19 to 0.67 +/- 0.19) over 5 days of culture. The negative force-frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 +/- 8 to 105 +/- 11 pF) and membrane currents were also reduced (I(Ca,L), from 3.98 +/- 0.39 to 2.12 +/- 0.37 pA pF; and I(K1), from 34.34p +/- 2.31 to 18.00 +/- 5.97 pA pF(-1)). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 +/- 2.5 to 34.2 +/- 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.

  5. Homophobia, Transphobia and Culture: Deconstructing Heteronormativity in English Primary Schools

    ERIC Educational Resources Information Center

    DePalma, Renee; Jennett, Mark

    2010-01-01

    This article presents some of the advances in legal support for addressing homophobia and transphobia in school settings and provides a critique of school-based policies that focus on these phenomena as particular incidents involving bullies and victims. Defining heteronormativity as a cultural phenomenon underpinning recognisable acts of…

  6. Homophobia, Transphobia and Culture: Deconstructing Heteronormativity in English Primary Schools

    ERIC Educational Resources Information Center

    DePalma, Renee; Jennett, Mark

    2010-01-01

    This article presents some of the advances in legal support for addressing homophobia and transphobia in school settings and provides a critique of school-based policies that focus on these phenomena as particular incidents involving bullies and victims. Defining heteronormativity as a cultural phenomenon underpinning recognisable acts of…

  7. Pyrethroid insecticide accumulation in primary cultures of cortical neurons in vitro

    EPA Science Inventory

    Primary cultures of neurons have been widely utilized to study the actions of pyrethroids and other neurotoxicants, with the presumption that the media concentration accurately reflects the dose received by the cells. However, recent studies have demonstrated that lipophilic comp...

  8. Establishing and maintaining primary cell cultures derived from the ctenophore Mnemiopsis leidyi.

    PubMed

    Vandepas, Lauren E; Warren, Kaitlyn J; Amemiya, Chris T; Browne, William E

    2017-04-01

    We have developed an efficient method for the preparation and maintenance of primary cell cultures isolated from adult Mnemiopsis leidyi, a lobate ctenophore. Our primary cell cultures are derived from tissue explants or enzymatically dissociated cells, and maintained in a complex undefined ctenophore mesogleal serum. These methods can be used to isolate, maintain and visually monitor ctenophore cells to assess proliferation, cellular morphology and cell differentiation in future studies. Exemplar cell types that can be easily isolated from primary cultures include proliferative ectodermal and endodermal cells, motile amebocyte-like cells, and giant smooth muscle cells that exhibit inducible contractile properties. We have also derived 'tissue envelopes' containing sections of endodermal canal surrounded by mesoglea and ectoderm that can be used to monitor targeted cell types in an in vivo context. Access to efficient and reliably generated primary cell cultures will facilitate the analysis of ctenophore development, physiology and morphology from a cell biological perspective.

  9. Toward a world-class culture in primary care: listening to the voice of the customer.

    PubMed

    Benson, D S

    1996-01-01

    This article on creating a world-class culture in primary care stresses the importance of customers. The concept of patients as external customers is addressed; the emphasis, however, is on healthcare professionals' responsibilities toward one another as internal customers.

  10. Correction: Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    PubMed

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-09-01

    Correction for 'Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics' by Robert A. Colvin et al., Metallomics, 2015, 7, 1111-1123.

  11. Pyrethroid insecticide accumulation in primary cultures of cortical neurons in vitro

    EPA Science Inventory

    Primary cultures of neurons have been widely utilized to study the actions of pyrethroids and other neurotoxicants, with the presumption that the media concentration accurately reflects the dose received by the cells. However, recent studies have demonstrated that lipophilic comp...

  12. Comparison of bacterial cultures of the larynx between dogs with laryngeal paralysis and normal dogs.

    PubMed

    Ganjei, J; Langenbach, A; Watrous, G; Hodgson, J

    2016-11-01

    To document the most common types of bacteria isolated from the canine larynx and to compare isolates, degree of growth and susceptibility patterns between dogs with laryngeal paralysis and dogs with normal laryngeal function. Laryngeal swabs were collected from each patient and submitted for bacterial culture and susceptibility testing. Dogs with laryngeal paralysis (n=23) underwent a unilateral arytenoid lateralisation and control dogs (n=24) underwent an elective orthopaedic procedure. Results of the cultures were compared between groups. Bacterial organisms isolated from the larynx were similar to those normally found in the oropharynx, trachea and lungs. The most common bacteria isolated from the larynges of all dogs were Escherichia coli, Klebsiella species and Pasteurella species. Pure colonies were more commonly seen in dogs with laryngeal paralysis while mixed colonies were more commonly seen in control dogs. Antimicrobial resistance was similar between study and control dogs. The laryngeal flora appears to contain bacteria that are commonly isolated from the oropharynx, trachea and lungs. Differences in antimicrobial susceptibility were not identified between study and control dogs. © 2016 British Small Animal Veterinary Association.

  13. Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels.

    PubMed

    Rosenzweig, D H; Chicatun, F; Nazhat, S N; Quinn, T M

    2013-12-01

    Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However, standard culture techniques require cell passaging, leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes, grown either in CE culture or passaged twice on static silicone dishes (SS culture; comparable to standard methods), were seeded in dense collagen gels and cultured for 3 weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes, CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3 weeks in culture, correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition, yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3 weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties, making them more suitable than SS cultured cells for tissue engineering applications. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  14. Organizational culture predicts job satisfaction and perceived clinical effectiveness in pediatric primary care practices.

    PubMed

    Brazil, Kevin; Wakefield, Dorothy B; Cloutier, Michelle M; Tennen, Howard; Hall, Charles B

    2010-01-01

    In recent years, there has been a growing understanding that organizational culture is related to an organization's performance. However, few studies have examined organizational culture in medical group practices. The purpose of this study was to examine the relationship of organizational culture on provider job satisfaction and perceived clinical effectiveness in primary care pediatric practices. This cross-sectional study included 36 primary care pediatric practices located in Connecticut. There were 374 participants in this study, which included 127 clinicians and 247 nonclinicians. Office managers completed a questionnaire that recorded staff and practice characteristics; all participants completed the Organizational Culture Scale, a questionnaire that assessed the practice on four cultural domains (i.e., group, developmental, rational, and hierarchical), and the Primary Care Organizational Questionnaire that evaluated perceived effectiveness and job satisfaction. Hierarchical linear models using a restricted maximum likelihood estimation method were used to evaluate whether the practice culture types predicted job satisfaction and perceived effectiveness. Group culture was positively associated with both satisfaction and perceived effectiveness. In contrast, hierarchical and rational culture were negatively associated with both job satisfaction and perceived effectiveness. These relationships were true for clinicians, nonclinicians, and the practice as a whole. Our study demonstrates that practice culture is associated with job satisfaction and perceived clinical effectiveness and that a group culture was associated with high job satisfaction and perceived effectiveness.

  15. Family, Culture and Achievement in the Primary Schools of Botswana.

    ERIC Educational Resources Information Center

    Lynch, Patrick D.

    This paper traces the development of the educational system of Botswana, a small south African country of one million, emphasizing its democratic origins and customs, historical influences, social trends, and economic support. Changes in the educational system, especially in universal primary education, since its independence from Great Britain in…

  16. Vessel Caliber in Normal Tension and Primary Open Angle Glaucoma Eyes With Hemifield Damage.

    PubMed

    Rao, Aparna; Agarwal, Komal; Mudunuri, Harika; Padhy, Debananda; Roy, Avik K; Mukherjee, Sujoy

    2017-01-01

    The purpose of the study was to evaluate retinal vessel diameter in age-matched normal tension glaucoma (NTG) and primary open angle glaucoma (POAG) eyes with hemifield involvement. Fundus photographs of patients with hemifield defect, good visibility of retinal nerve fiber layer defect, and vessels were compared with 30 controls. One eye of each patient (28 NTG and 30 POAG) was randomly chosen for analysis using Image J software by different clinicians at 2 levels. The structural parameters analyzed included retinal nerve fiber layer defect width, diameter of vessels (superotemporal or inferotemporal artery and superotemporal or inferotemporal vein). The average superotemporal artery diameter was similar in NTG (71±16.8 µm), POAG (79±26.6 µm), and controls (82±14.1 µm), P=0.2 with similar pattern seen for other vessels. The affected quadrant in all eyes and those with disc hemorrhage (n=8) did not have significantly different arteries and veins diameter as compared with the unaffected quadrant in that eye. No relation of artery or vein diameter with retinal nerve fiber layer defect width or clinical variables in NTG or POAG eyes was seen on multivariate regression analysis. The vessel diameter was not significantly different in the affected and unaffected quadrants of age and severity-matched NTG and POAG eyes with hemifield structural and functional defect as reported in earlier studies. These results point toward the possibility that vascular diameter changes may not be the cause for glaucomatous changes.

  17. Clinical profiles of primary open angle glaucoma versus normal tension glaucoma patients: a pilot study.

    PubMed

    Asrani, Sanjay; Samuels, Brian; Thakur, Mugdha; Santiago, Cecilia; Kuchibhatla, Maragatha

    2011-05-01

    To study the health profiles and behavioral differences between primary open angle glaucoma (POAG) and normal tension glaucoma (NTG) patients. Confidential mail survey of ICD-9 defined POAG (n = 277) and NTG (n = 116) patients was carried out to collect information regarding age, gender, height, weight, waist circumference, blood pressure (BP), medications [prescription (Rx) and over the counter (OTC)], education, and the Short Health Anxiety Inventory (SHAI). NTG patients (59/116) were significantly more likely to respond to the survey than POAG patients (43/277), p = 0.0001, and to have obtained college level education (70% vs. 42%), p = 0.003. Body weight, waist circumference, body mass index, systolic BP, pulse pressure, and ratio of prescription medications to OTC medications were significantly lower in the NTG group (p < 0.0001). The SHAI score, mean age, and diastolic BP were not different between the two groups. A subgroup analysis of college-educated patients in both groups revealed persisting significant differences in waist circumference, systolic BP, and ratio of Rx to OTC medications. NTG patients do not have significantly greater health anxiety than POAG patients. NTG patients are more likely to have lower BMI, systolic BP, and ratio of Rx to OTC medications than POAG patients. The pilot nature of this study limits generalizability of these findings. Larger studies confirming these findings and elucidating the role of refractive error and medical history on such differences are needed.

  18. Mutation Patterns of 16 Genes in Primary and Secondary Acute Myeloid Leukemia (AML) with Normal Cytogenetics

    PubMed Central

    Fernandez-Mercado, Marta; Yip, Bon Ham; Pellagatti, Andrea; Davies, Carwyn; Larrayoz, María José; Kondo, Toshinori; Pérez, Cristina; Killick, Sally; McDonald, Emma-Jane; Odero, María Dolores; Agirre, Xabier; Prósper, Felipe; Calasanz, María José; Wainscoat, James S.; Boultwood, Jacqueline

    2012-01-01

    Acute myeloid leukemia patients with normal cytogenetics (CN-AML) account for almost half of AML cases. We aimed to study the frequency and relationship of a wide range of genes previously reported as mutated in AML (ASXL1, NPM1, FLT3, TET2, IDH1/2, RUNX1, DNMT3A, NRAS, JAK2, WT1, CBL, SF3B1, TP53, KRAS and MPL) in a series of 84 CN-AML cases. The most frequently mutated genes in primary cases were NPM1 (60.8%) and FLT3 (50.0%), and in secondary cases ASXL1 (48.5%) and TET2 (30.3%). We showed that 85% of CN-AML patients have mutations in at least one of ASXL1, NPM1, FLT3, TET2, IDH1/2 and/or RUNX1. Serial samples from 19 MDS/CMML cases that progressed to AML were analyzed for ASXL1/TET2/IDH1/2 mutations; seventeen cases presented mutations of at least one of these genes. However, there was no consistent pattern in mutation acquisition during disease progression. This report concerns the analysis of the largest number of gene mutations in CN-AML studied to date, and provides insight into the mutational profile of CN-AML. PMID:22912701

  19. Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

    PubMed Central

    Ryu, Hyun Mi; Park, Sung Gyoo; Yea, Sung Su; Jang, Won Hee; Yang, Young-Il; Jung, Guhung

    2006-01-01

    AIM: To find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray. METHODS: Primary normal human hepatocytes (PNHHs) were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS: From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were transfected with pHBV1.2×, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV-mediated NF-κB activation. CONCLUSION: During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK. PMID:16937494

  20. Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells

    SciTech Connect

    Rettig, W.F.; Garin-Chesa, P.; Beresford, H.R.; Oettgen, H.F.; Melamed, M.R.; Old, L.J.

    1988-05-01

    Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, the authors have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and > 200 tumor specimens. Differential patterns of F19, F24, G171, G253, S5, and Thy-1 antigen expression were found to characterize (i) subsets of cultured sarcoma cell lines, (ii) cultured fibroblasts derived from various organs, (iii) normal resting and activated mesenchymal tissues, and (iv) sarcoma and nonmesenchymal tumor tissues. These results provide a basic surface antigenic map for cultured mesenchymal cells and mesenchymal tissues and permit the classification of human sarcomas according to their antigenic phenotypes.

  1. Adherent Primary Cultures of Mouse Intercostal Muscle Fibers for Isolated Fiber Studies

    PubMed Central

    Robison, Patrick; Hernández-Ochoa, Erick O.; Schneider, Martin F.

    2011-01-01

    Primary culture models of single adult skeletal muscle fibers dissociated from locomotor muscles adhered to glass coverslips are routine and allow monitoring of functional processes in living cultured fibers. To date, such isolated fiber cultures have not been established for respiratory muscles, despite the fact that dysfunction of core respiratory muscles leading to respiratory arrest is the most common cause of death in many muscular diseases. Here we present the first description of an adherent culture system for single adult intercostal muscle fibers from the adult mouse. This system allows for monitoring functional properties of these living muscle fibers in culture with or without electrical field stimulation to drive muscle fiber contraction at physiological or pathological respiratory firing patterns. We also provide initial characterization of these fibers, demonstrating several common techniques in this new model system in the context of the established Flexor Digitorum Brevis muscle primary culture model. PMID:21869860

  2. A Multi-Cultural Interaction through Video Conferencing in Primary Schools

    ERIC Educational Resources Information Center

    Duygu Eristi, Suzan

    2012-01-01

    This study investigated Turkish and Canadian primary school students' ways of expressing their perception of cultural understanding through video conferencing and that of cultural interaction through video conferencing. The qualitative research data were collected in the form of interviews. The results obtained were analyzed and interpreted based…

  3. Creative Partnerships? Cultural Policy and Inclusive Arts Practice in One Primary School

    ERIC Educational Resources Information Center

    Hall, Christine; Thomson, Pat

    2007-01-01

    This article traces the "cultural turn" in UK educational policy through an analysis of the Creative Partnerships policy (New Labour's "flagship programme in the cultural education field") and a consideration of an arts project funded under this initiative in one primary school. It argues that current educational policy…

  4. Contribution to Cultural Organization, Working Motivation and Job Satisfaction on the Performance of Primary School Teacher

    ERIC Educational Resources Information Center

    Murtedjo; Suharningsih

    2016-01-01

    The purposes of this study are: (1) describes the performance of the teacher, organizational culture, work motivation and job satisfaction; (2) determine whether there is a significant direct relationship between organizational culture, work motivation and job satisfaction on the performance of primary school teachers. Through the study of the…

  5. English and French Pedagogical Cultures: Convergence and Divergence in Cameroonian Primary School Teachers' Discourse

    ERIC Educational Resources Information Center

    Esch, Edith

    2012-01-01

    This article approaches the phenomenon of the continuing influence of French and English pedagogical cultures in Africa relying on post-modern notions of time and space. It reports on a project carried out in Cameroon where both cultures are in contact and where the teachers from two primary schools were observed and interviewed over a period of…

  6. A qualitative study of the cultural changes in primary care organisations needed to implement clinical governance.

    PubMed

    Marshall, Martin; Sheaff, Rod; Rogers, Anne; Campbell, Stephen; Halliwell, Shirley; Pickard, Susan; Sibbald, Bonnie; Roland, Martin

    2002-08-01

    It is commony claimed that changing the culture of health organisations is a fundamental prerequisite for improving the National Health Service (NHS). Little is currently known about the nature or importance of culture and cultural change in primary care groups and trusts (PCG/Ts) or their constituent general practices. To investigate the importance of culture and cultural change for the implementation of clinical governance in general practice by PCG/Ts, to identify perceived desirable and undesirable cultural attributes of general practice, and to describe potential facilitators and barriers to changing culture. Qualitative: case studies using data derived from semi-structured interviews and review of documentary evidence. Fifty senior non-clinical and clinical managers from 12 purposely sampled PCGs or trusts in England. Senior primary care managers regard culture and cultural change as fundamental aspects of clinical governance. The most important desirable cultural traits were the value placed on a commitment to public accountability by the practices, their willingness to work together and learn from each other, and the ability to be self-critical and learn from mistakes. The main barriers to cultural change were the high level of autonomy of practices and the perceived pressure to deliver rapid measurable changes in general practice. The culture of general practice is perceived to be an important component of health system reform and quality improvement. This study develops our understanding of a changing organisational culture in primary care; however, further work is required to determine whether culture is a useful practical lever for initiating or managing improvement.

  7. A qualitative study of the cultural changes in primary care organisations needed to implement clinical governance.

    PubMed Central

    Marshall, Martin; Sheaff, Rod; Rogers, Anne; Campbell, Stephen; Halliwell, Shirley; Pickard, Susan; Sibbald, Bonnie; Roland, Martin

    2002-01-01

    BACKGROUND: It is commony claimed that changing the culture of health organisations is a fundamental prerequisite for improving the National Health Service (NHS). Little is currently known about the nature or importance of culture and cultural change in primary care groups and trusts (PCG/Ts) or their constituent general practices. AIMS: To investigate the importance of culture and cultural change for the implementation of clinical governance in general practice by PCG/Ts, to identify perceived desirable and undesirable cultural attributes of general practice, and to describe potential facilitators and barriers to changing culture. DESIGN: Qualitative: case studies using data derived from semi-structured interviews and review of documentary evidence. SETTING: Fifty senior non-clinical and clinical managers from 12 purposely sampled PCGs or trusts in England. RESULTS: Senior primary care managers regard culture and cultural change as fundamental aspects of clinical governance. The most important desirable cultural traits were the value placed on a commitment to public accountability by the practices, their willingness to work together and learn from each other, and the ability to be self-critical and learn from mistakes. The main barriers to cultural change were the high level of autonomy of practices and the perceived pressure to deliver rapid measurable changes in general practice. CONCLUSIONS: The culture of general practice is perceived to be an important component of health system reform and quality improvement. This study develops our understanding of a changing organisational culture in primary care; however, further work is required to determine whether culture is a useful practical lever for initiating or managing improvement. PMID:12171222

  8. Establishment of a Novel Model for Anticancer Drug Resistance in Three-Dimensional Primary Culture of Tumor Microenvironment

    PubMed Central

    Sakurai, Masashi; Enjoji, Shuhei; Umata, Koji; Fujiwara, Nobuyuki; Tsuji, Shunya; Hazama, Shoichi

    2016-01-01

    Tumor microenvironment has been implicated in tumor development and progression. As a three-dimensional tumor microenvironment model, air liquid interface (ALI) organoid culture from oncogene transgenic mouse gastrointestinal tissues was recently produced. However, ALI organoid culture system from tissues of colorectal cancer patients has not been established. Here, we developed an ALI organoid model from normal and tumor colorectal tissues of human patients. Both organoids were successfully generated and showed cystic structures containing an epithelial layer and surrounding mesenchymal stromal cells. Structures of tumor organoids closely resembled primary tumor epithelium. Expression of an epithelial cell marker, E-cadherin, a goblet cell marker, MUC2, and a fibroblast marker, vimentin, but not a myofibroblast marker, α-smooth muscle actin (SMA), was observed in normal organoids. Expression of E-cadherin, MUC2, vimentin, and α-SMA was observed in tumor organoids. Expression of a cancer stem cell marker, LGR5 in tumor organoids, was higher than that in primary tumor tissues. Tumor organoids were more resistant to toxicity of 5-fluorouracil and Irinotecan than colorectal cancer cell lines, SW480, SW620, and HCT116. These findings indicate that ALI organoid culture from colorectal cancer patients may become a novel model that is useful for examining resistance to chemotherapy in tumor microenvironment. PMID:28119740

  9. The Effect of Organizational Trust on the Culture of Teacher Leadership in Primary Schools

    ERIC Educational Resources Information Center

    Demir, Kamile

    2015-01-01

    The purpose of this research is to examine the effect of the level of trust of primary school teachers towards their organization in relation to their perceptions of the school having a culture of teacher leadership. Participants of the study consisted of 378 teachers working in Burdur public primary schools. The data collection tool used two…

  10. Turkish Primary School Teachers' Perceptions of School Culture Regarding ICT Integration

    ERIC Educational Resources Information Center

    Tezci, Erdogan

    2011-01-01

    The current study aimed at identifying Turkish primary school teachers' perceptions of school culture regarding ICT integration in education. In addition, the current study was designed to investigate factors that might influence their perceptions. The participants were 1540 primary school teachers. The findings revealed that the teachers'…

  11. Turkish Primary School Teachers' Perceptions of School Culture Regarding ICT Integration

    ERIC Educational Resources Information Center

    Tezci, Erdogan

    2011-01-01

    The current study aimed at identifying Turkish primary school teachers' perceptions of school culture regarding ICT integration in education. In addition, the current study was designed to investigate factors that might influence their perceptions. The participants were 1540 primary school teachers. The findings revealed that the teachers'…

  12. Meaningful Cultural Learning by Imitative Participation: The Case of Abstract Thinking in Primary School

    ERIC Educational Resources Information Center

    van Oers, Bert

    2012-01-01

    The article describes a theory-driven approach to meaningful learning in primary schools, based on the Vygotskian cultural-historical theory of human development and learning. This approach is elaborated into an educational concept called "developmental education" that is implemented in the Netherlands in many primary schools. In this…

  13. Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research.

    PubMed

    Ray, Balmiki; Chopra, Nipun; Long, Justin M; Lahiri, Debomoy K

    2014-09-16

    Culturing primary cortical neurons is an essential neuroscience technique. However, most cultures are derived from rodent brains and standard protocols for human brain cultures are sparse. Herein, we describe preparation, maintenance and major characteristics of a primary human mixed brain culture, including neurons, obtained from legally aborted fetal brain tissue. This approach employs standard materials and techniques used in the preparation of rodent neuron cultures, with critical modifications. This culture has distinct differences from rodent cultures. Specifically, a significant numbers of cells in the human culture are derived from progenitor cells, and the yield and survival of the cells grossly depend on the presence of bFGF. In the presence of bFGF, this culture can be maintained for an extended period. Abundant productions of amyloid-β, tau and proteins make this a powerful model for Alzheimer's research. The culture also produces glia and different sub-types of neurons. We provide a well-characterized methodology for human mixed brain cultures useful to test therapeutic agents under various conditions, and to carry forward mechanistic and translational studies for several brain disorders.

  14. Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

    PubMed

    Orsini, Malina; Sperber, Saskia; Noor, Fozia; Hoffmann, Esther; Weber, Susanne N; Hall, Rabea A; Lammert, Frank; Heinzle, Elmar

    2017-06-08

    Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 9999: 1-8, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Primary outgrowth cultures are a reliable source of human pancreatic stellate cells.

    PubMed

    Han, Song; Delitto, Daniel; Zhang, Dongyu; Sorenson, Heather L; Sarosi, George A; Thomas, Ryan M; Behrns, Kevin E; Wallet, Shannon M; Trevino, Jose G; Hughes, Steven J

    2015-11-01

    Recent advances demonstrate a critical yet poorly understood role for the pancreatic stellate cell (PSC) in the pathogenesis of chronic pancreatitis (CP) and pancreatic cancer (PC). Progress in this area has been hampered by the availability, fidelity, and/or reliability of in vitro models of PSCs. We examined whether outgrowth cultures from human surgical specimens exhibited reproducible phenotypic and functional characteristics of PSCs. PSCs were cultured from surgical specimens of healthy pancreas, CP and PC. Growth dynamics, phenotypic characteristics, soluble mediator secretion profiles and co-culture with PC cells both in vitro and in vivo were assessed. Forty-seven primary cultures were established from 52 attempts, demonstrating universal α-smooth muscle actin and glial fibrillary acidic protein but negligible epithelial surface antigen expression. Modification of culture conditions consistently led to cytoplasmic lipid accumulation, suggesting induction of a quiescent phenotype. Secretion of growth factors, chemokines and cytokines did not significantly differ between donor pathologies, but did evolve over time in culture. Co-culture of PSCs with established PC cell lines resulted in significant changes in levels of multiple secreted mediators. Primary PSCs co-inoculated with PC cells in a xenograft model led to augmented tumor growth and metastasis. Therefore, regardless of donor pathology, outgrowth cultures produce PSCs that demonstrate consistent growth and protein secretion properties. Primary cultures from pancreatic surgical specimens, including malignancies, may represent a reliable source of human PSCs.

  16. Impact of Different Normality Thresholds for 24-hour ABPM at the Primary Health Care Level

    PubMed Central

    Grezzana, Guilherme Brasil; Moraes, David William; Stein, Airton Tetelbon; Pellanda, Lucia Campos

    2017-01-01

    Background Hypertension is an important risk factor for cardiovascular outcomes. Primary health care (PHC) physicians should be prepared to act appropriately in the prevention of cardiovascular risk factors. However, the rates of patients with control of blood pressure (BP) remain low. The impact of the reclassification of high BP by 24-hour ambulatory BP monitoring (ABPM) can lead to different medical decisions in PHC. Objective To evaluate the agreement between the BP measured by a conventional method by PHC physicians and by 24-hour ABPM, considering different BP normal thresholds for the 24-hour ABPM according to the V Brazilian ABPM Guidelines and the European Society of Hypertension Guidelines. Methods A cross-sectional study including 569 hypertensive patients. The BP was initially measured by the PHC physicians and, later, by 24-hour ABPM. The BP measurements were obtained independently between the two methods. The therapeutic targets for the conventional BP followed the guidelines by the Eighth Joint National Committee (JNC 8), the V ABPM Brazilian Guidelines, and the 2013 European Hypertension Guidelines. Results There was an accuracy of 54.8% (95% confidence interval [95%CI] 0.51 - 0.58%) for the BP measured with the conventional method when compared with the 24-hour ABPM, with a sensitivity of 85% (95%CI 80.8 - 88.6%), specificity of 31.9% (95%CI 28.7 - 34.7%), and kappa value of 0.155, when considering the European Hypertension Guidelines. When using more stringent thresholds to characterize the BP as "normal" by ABPM, the accuracy was 45% (95%CI 0.41 - 0.47%) for conventional measurement when compared with 24-hour ABPM, with a sensitivity of 86.7% (95%CI 0.81 - 0.91%), specificity of 29% (95%CI 0.26 - 0.30%), and kappa value of 0.103. Conclusion The BP measurements obtained by PHC physicians showed low accuracy when compared with those obtained by 24-hour ABPM, regardless of the threshold set by the different guidelines. PMID:28099585

  17. Functional gait outcomes for idiopathic normal pressure hydrocephalus after primary endoscopic third ventriculostomy.

    PubMed

    Sankey, Eric W; Jusué-Torres, Ignacio; Elder, Benjamin D; Goodwin, C Rory; Batra, Sachin; Hoffberger, Jamie; Lu, Jennifer; Blitz, Ari M; Rigamonti, Daniele

    2015-08-01

    We evaluated if patients with idiopathic normal pressure hydrocephalus (iNPH) showed functional improvement after primary endoscopic third ventriculostomy (ETV). The efficacy of ETV for iNPH remains controversial. We retrospectively reviewed 10 consecutive patients treated between 2009 and 2011 with ETV for iNPH. Seven patients with a median age of 73 years (range: 60-80) who underwent a primary ETV for iNPH were included for analysis. Median follow-up was 39 months (range: 26-46). Post-ETV stoma and aqueductal and cisternal flows were confirmed via high resolution, gradient echo and phase contrast MRI. Post-ETV timed up and go (TUG) and Tinetti performance oriented mobility assessment scores were compared to pre- and post-lumbar puncture (LP) values. A second LP was performed if ETV failed to sustain the observed improvement after initial LP. Patients who demonstrated ETV failure were subsequently shunted. Compared to pre-LP TUG and Tinetti values of 14.00 seconds (range: 12.00-23.00) and 22 (range: 16-24), post-LP scores improved to 11.00 seconds (range: 8.64-15.00; p=0.06) and 25 (range: 24-28; p=0.02), respectively. ETV failed to sustain this improvement with slight worsening between pre-LP and post-ETV TUG and Tinetti scores. Improvement from pre-LP assessment was regained after shunting and at last follow-up with TUG and Tinetti scores of 12.97 seconds (range: 9.00-18.00; p=0.250) and 25 (range: 18-27; p=0.07), and 11.87 seconds (range: 8.27-18.50; p=0.152) and 23 (range: 18-26; p=0.382), respectively. Despite stoma patency, ETV failed to sustain functional improvement seen after LP, however, improvement was regained after subsequent shunting suggesting that shunt placement remains the preferred treatment for iNPH.

  18. Characterization of benzodiazepine receptors in primary cultures of fetal mouse brain and spinal cord neurons.

    PubMed

    Huang, A; Barker, J L; Paul, S M; Moncada, V; Skolnick, P

    1980-05-26

    Primary cultures of fetal mouse brain and spinal cord were examined for the presence of binding sites for [3H]diazepam. Both brain and spinal cord cultures contain high affinity binding sites which resemble benzodiazepine receptors found in mammalian CNS with respect to both pharmacologic profile and response to exogenously applied GABA. These observations, coupled with the electrophysiologic properties of these cells suggest that primary cultures of fetal mouse brain and spinal cord may be valid models for studying the role and regulation of the benzodiazepine receptor.

  19. Cryopreservation and lentiviral-mediated genetic modification of human primary cultured corneal endothelial cells.

    PubMed

    Suh, Leejee H; Zhang, Cheng; Chuck, Roy S; Stark, Walter J; Naylor, Stuart; Binley, Katie; Chakravarti, Shukti; Jun, Albert S

    2007-07-01

    To determine the viability and potential usefulness of cryopreserved human primary cultured corneal endothelial cells by characterizing their morphology, gene expression, and ability for genetic modification by the lentiviral vector equine infectious anemia virus (EIAV). Primary cultured endothelial cells were dissociated from human corneas and grown in organ culture medium. Corneal endothelial cell origin was confirmed by morphology and immunostaining with polyclonal anti-collagen VIII antibodies. Cells of different passages were cryopreserved in medium containing dimethyl sulfoxide and were assessed after thawing for morphology, proliferative capacity, gene expression, and ability to form cell-cell junctions. EIAV encoding enhanced green fluorescent protein (eGFP) was used to transduce cryopreserved human corneal endothelial cells. Transduced cells were then sorted by fluorescence-activated cell sorting (FACS) and imaged with fluorescence microscopy. Cryopreserved, primary, cultured human corneal endothelial cells are viable and retain their ability to proliferate, produce collagen VIII, and express ZO-1, a tight-junction protein. EIAV-based gene transfer of eGFP is highly efficient and nontoxic to cryopreserved human primary cultured corneal endothelial cells. These genetically modified cells can be selected to nearly pure populations with FACS sorting. Human primary cultured corneal endothelial cells retain their phenotypic properties after cryopreservation. The ability to store, genetically modify, and sort these cells through FACS to pure populations has the potential to greatly expand their future therapeutic application to treat corneal endothelial disorders.

  20. Comparison of Prelaminar Thickness between Primary Open Angle Glaucoma and Normal Tension Glaucoma Patients

    PubMed Central

    Jung, Youn Hea; Park, Hae-Young L.; Jung, Kyoung In; Park, Chan Kee

    2015-01-01

    Main Objective The thinning of prelaminar tissue and prelamina cupping is known to occur by ischemia, as we see in anterior ischemic optic neuropathy. Since normal tension glaucoma (NTG) is thought to be more related to vascular factor than in primary open-angle glaucoma (POAG), we hypothesized that prelamina thinning may occur prominently in NTG patients. This study investigated the difference in prelaminar tissue thickness between patients with POAG and NTG and verified the factors related to prelaminar thinning. Methods Complete ophthalmic examination including standard automatic perimetry was performed in all patients. The prelaminar tissue thickness was measured in all patients by performing enhanced depth imaging with a Heidelberg Spectralis Optical Coherence Tomography. The retinal nerve fiber layer and optic nerve head parameters were obtained using the Heidelberg Retina Tomography II and Cirrus Optical Coherence Tomography. Various ocular factors and their relationships with prelaminar thickness were analyzed. Results The mean prelaminar tissue thickness was significantly thinner in patients with POAG than in those with NTG. The difference in the prelaminar thickness between patients with POAG and those with NTG was greater in the early field defect group than in the moderate and severe field groups. In multivariate analysis, the mean prelaminar thickness was related to the intraocular pressure, mean deviation, cup-disc ratio, and cup volume. Conclusions The prelaminar tissue was thinner in patients with POAG than in patients with NTG, and intraocular pressure had a strong influence on the prelaminar thickness in both POAG and NTG. This may indicate that mechanical compression is the main pathogenic factor in both POAG and NTG. PMID:25793734

  1. B Cell Depletion Therapy Normalizes Circulating Follicular Th Cells in Primary Sjögren Syndrome.

    PubMed

    Verstappen, Gwenny M; Kroese, Frans G M; Meiners, Petra M; Corneth, Odilia B; Huitema, Minke G; Haacke, Erlin A; van der Vegt, Bert; Arends, Suzanne; Vissink, Arjan; Bootsma, Hendrika; Abdulahad, Wayel H

    2017-01-01

    To assess the effect of B cell depletion therapy on effector CD4+ T cell homeostasis and its relation to objective measures of disease activity in patients with primary Sjögren syndrome (pSS). Twenty-four patients with pSS treated with rituximab (RTX) and 24 healthy controls (HC) were included. Frequencies of circulating effector CD4+ T cell subsets were examined by flow cytometry at baseline and 16, 24, 36, and 48 weeks after the first RTX infusion. Th1, Th2, follicular Th (TFH), and Th17 cells were discerned based on surface marker expression patterns. Additionally, intracellular cytokine staining was performed for interferon-γ, interleukin (IL)-4, IL-21, and IL-17 and serum levels of these cytokines were analyzed. In patients with pSS, frequencies of circulating TFH cells and Th17 cells were increased at baseline compared with HC, whereas frequencies of Th1 and Th2 cells were unchanged. B cell depletion therapy resulted in a pronounced decrease in circulating TFH cells, whereas Th17 cells were only slightly lowered. Frequencies of IL-21-producing and IL-17-producing CD4+ T cells and serum levels of IL-21 and IL-17 were also reduced. Importantly, the decrease in circulating TFH cells was associated with lower systemic disease activity over time, as measured by the European League Against Rheumatism Sjögren's Syndrome Disease Activity Index scores and serum IgG levels. B cell depletion therapy in patients with pSS results in normalization of the elevated levels of circulating TFH cells. This reduction is associated with improved objective clinical disease activity measures. Our observations illustrate the pivotal role of the crosstalk between B cells and TFH cells in the pathogenesis of pSS.

  2. Establishment of a primary culture of polymorphous low grade adenocarcinoma cells.

    PubMed

    Teixeira, Lucas Novaes; Montalli, Victor Angelo Martins; de Oliveira, Silvia Borges Pimentel; Toledo, Thais Fernanda Santos; Martinez, Elizabeth Ferreira; de Araújo, Vera Cavalcanti

    2017-10-01

    The aim of the present study was to establish a primary cell culture derived from polymorphous low grade adenocarcinoma (PLGA). The neoplastic cells were derived from a 57-year-old female patient diagnosed with PLGA. A fragment of the tumor was collected and submitted to enzymatic digestion followed by centrifugation on a Percoll gradient. The cell population was characterized by means of immunofluorescence and detection of PRKD1 gene mutations. Epifluorescence analysis of the primary culture revealed that the malignant epithelial cells were predominantly polygonal in shape and positive for cytokeratin 7, vimentin, and S100. The doubling time of the cell culture was 86.73h. The restriction digestion assay showed that the neoplastic cells possess PRKD1 gene mutations. The establishment of primary cell culture derived from PLGA should be considered a useful tool for molecular analysis of this salivary gland tumor. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Ultrastructural and biochemical alterations induced in human, rat and mouse hepatocytes in primary culture exposed to selected carcinogens

    SciTech Connect

    Cole, K.H.

    1987-01-01

    Aflatoxin B1 (AFB{sub 1}), dimethylnitrosamine (DMN), 2-acetylaminofluorene (2-AAF) and actinomycin D are all potential human liver carcinogens. In order to investigate carcinogenic susceptibility of human liver to these agents, primary cultures of normal human hepatocytes were exposed to the four carcinogens. In the first series of experiments, human, rat, and mouse hepatocytes in primary culture were exposed to actinomycin D, AFB{sub 1}, and DMN for 24 h and examined for ultrastructural alterations. In an effort to relate the ultrastructural effects with total covalent binding of the carcinogen to DNA, human, rat and mouse hepatocytes were exposed to 2.0 {times} 10{sup {minus}7} M ({sup 3}H)AFB{sub 1} for 24 h. Hepatocytes from male rats had the highest degree of ({sup 3}H)AFB{sub 1}-DNA binding. Human hepatocytes contained the next highest binding level, while hepatocytes from female rats bound 38 pmoles/mg DNA. The AFB{sub 1}-DNA binding level in mouse hepatocytes was 1.4 pmoles/mg DNA. In similar experiments, human, and male and female rat hepatocytes in primary culture were exposed to the carcinogen 2-acetylamino (9-{sup 14}C)fluorene for 24 h. It was determined that male rat hepatocytes had the highest amount of radiolabeled 2-AAF bound to their DNA, female rats contained 0.57 nmoles/mg DNA, while human hepatocytes contained 0.29 nmoles/mg DNA.

  4. Response to hypogravity of normal in vitro cultured follicular cells from thyroid

    NASA Astrophysics Data System (ADS)

    Meli, Antonella; Perrella, Giuseppina; Curcio, Francesco; Saverio, F.; Impiombato, Ambesi

    Aim of this investigation is the study of molecular modifications occurring in differentiated mammalian cells exposed to gravitational changes. The test system chosen is a well characterized clone of differentiated, normal thyroid follicular cells (FRTL5) in long-term culture. As a follow-up to our recent experiment performed during the MASER-7 sounding rocket mission, flown for European Space Agency by Swedish Space Corporation in May 1996, we evaluated FRTL5 cells responses to Thyroid Stimulating Hormone dependent cAMP production under acute hypogravity conditions obtained in a fast rotating clinostat. Following this approach, we evaluated the FRTL5 cells response to TSH under microgravity conditions in order to optimize experimental tools and strategies in preparation to, and in between real flight missions.

  5. An improved protocol for primary culture of cardiomyocyte from neonatal mice.

    PubMed

    Sreejit, P; Kumar, Suresh; Verma, Rama S

    2008-01-01

    The primary culture of neonatal mice cardiomyocyte model enables researchers to study and understand the morphological, biochemical, and electrophysiological characteristics of the heart, besides being a valuable tool for pharmacological and toxicological studies. Because cardiomyocytes do not proliferate after birth, primary myocardial culture is recalcitrant. The present study describes an improved method for rapid isolation of cardiomyocytes from neonatal mice, as well as the maintenance and propagation of such cultures for the long term. Immunocytochemical and gene expression data also confirmed the presence of several cardiac markers in the beating cells during the long-term culture condition used in this protocol. The whole culture process can be effectively shortened by reducing the enzyme digestion period and the cardiomyocyte enrichment step.

  6. Investigating the establishment of primary cell culture from different abalone (Haliotis midae) tissues

    PubMed Central

    Auzoux-Bordenave, Stéphanie; Niesler, Carola; Roodt-Wilding, Rouvay

    2010-01-01

    The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level. PMID:20680682

  7. Investigating the establishment of primary cell culture from different abalone (Haliotis midae) tissues.

    PubMed

    van der Merwe, Mathilde; Auzoux-Bordenave, Stéphanie; Niesler, Carola; Roodt-Wilding, Rouvay

    2010-07-01

    The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level.

  8. Novel approach of signal normalization for depth profile of cultural heritage materials

    NASA Astrophysics Data System (ADS)

    Syvilay, D.; Detalle, V.; Wilkie-Chancellier, N.; Texier, A.; Martinez, L.; Serfaty, S.

    2017-01-01

    The investigation of cultural heritage materials is always complex and specific because unique. Materials are most often heterogeneous and organized in several layers such as mural paintings or corrosion products. The characterization of a complete artwork's stratigraphy is actually one of the questions of science conservation. Indeed, the knowledge of these layers allows completing the history of the work of art and a better understanding of alteration processes in order to set up an appropriate conservation action. The LIBS technique has been employed to study the stratigraphy of an artwork thanks to the ablation laser. However, as we know, atomic information could be insufficient to characterize two materials composed by the same based elements. Therefore, an additional molecular analysis, like Raman spectroscopy; is sometimes necessary for a better identification of the material in particular for organic coatings in cultural heritage. We suggest in this study to use Standard Normal Variate (SNV) as a common normalization for different kinds of spectra (LIBS and Raman spectroscopy) combined with a 3D colour representation for stratigraphic identification of the different layers composing the complex material from artwork. So in this investigation, the SNV method will be applied on LIBS and Raman spectra but also on baseline Raman spectra often considering as nuisance. The aim of this study is to demonstrate the versatility of SNV applied on varied spectra like LIBS, Raman spectra as well as the luminescence background. This original work considers the SNV with a 3D colour representation as a probable new perspective for an easy recognition of a structure layered with a direct overview of the depth profile of the artwork.

  9. Effects of phenobarbital on aniline metabolism in primary liver cell culture of rats with ethionine-induced liver disorder.

    PubMed

    Noguchi, M; Nitoh, S; Mabuchi, M; Kawai, Y

    1996-04-01

    In experiment 1, the amount of aniline (AN) metabolites in the primary cell culture medium of the liver cells obtained from ethionine (ET)-treated rats was compared with that of the control (normal) rats. Although the metabolites detected in both groups were p-aminophenol (p-AP), N-acetyl-p-AP (AAP), acetoanilide (AAN), AAP-glucuronide (AAPG), phenylhydroxylamine sulfate (PHAS) and p-AP-glucuronide (p-APG), the amount of AAP was lower and that of p-APG was markedly higher in the ET-treated rats than in the control rats. In experiment 2, phenobarbital (PB) was orally administered to the ET-treated and control rats at a dose of 100 mg/kg. The time course changes in AN metabolites in the primary cell culture medium of liver cells obtained at 2 or 48 hr after PB treatment were compared with those without PB treatment. In the ET-treated rats, the amount of PHAS was slightly higher at 2 hr after PB treatment, and that of AAP was lower and that of p-APG was higher at 48 hr after PB treatment as compared with those without PB treatment. In the control rats, the amounts of AAP, AAN, p-AP and p-APG at 2 hr after PB treatment remained lower than those without PB treatment, and that of AAP was markedly lower and that of p-APG was higher at 48 hr after PB treatment as compared with those without PB treatment. These findings indicated greater detoxication in the primary liver cell culture in the ET-treated rats than in the control rats. Furthermore, detoxication was greater in the primary cell culture of liver cell obtained from the ET-treated rats after PB treatment than from those without PB treatment, because the production of acetylates (AAP) decreased and p-APG increased (induction of conjugated enzyme) in the PB treatment group.

  10. Growth sensitivity of a recombinant simian virus 5 P/V mutant to type I interferon differs between tumor cell lines and normal primary cells.

    PubMed

    Wansley, Elizabeth K; Dillon, Patrick J; Gainey, Maria D; Tam, James; Cramer, Scott D; Parks, Griffith D

    2005-04-25

    A paramyxovirus SV5 mutant (rSV5-P/V-CPI-) that encodes 6 naturally-occurring P/V gene substitutions is a potent inducer of type I interferon (IFN) and is restricted for low moi growth, two phenotypes not seen with WT SV5. In this study, we have compared the IFN sensitivity of WT SV5 and the rSV5-P/V-CPI- mutant in tumor cell lines and in cultures of normal primary cells. We have tested the hypothesis that differences in IFN induction elicited by WT rSV5 and rSV5-P/V-CPI- are responsible for differences in low moi growth and spread. In contrast to WT SV5, low moi infection of A549 lung carcinoma cells with rSV5-P/V-CPI- resulted in a plateau of virus production by 24-48 h pi when secreted IFN levels were between approximately 100 and 1000 U/ml. Gene microarray and RT-PCR analyses identified IFN genes and IFN-stimulated genes whose expression were increased by infection of A549 cells with WT and P/V mutant viruses. Restricted low moi growth and spread of rSV5-P/V-CPI- in A549 cells was relieved in the presence of neutralizing antibodies to IFN-beta but not TNF-alpha. When A549 or MDA-MB-435 breast tumor cells were pretreated with IFN, both WT and P/V mutant viruses showed delayed spread and approximately 10-fold reduction in virus yield, but infections were not eliminated. Using normal primary human epithelial cells that have undergone limited passage in culture, WT rSV5 and rSV5-P/V-CPI- displayed high moi growth properties that were similar to that seen in A549 cells. However, IFN pretreatment of these primary cells as well as normal human lung cells eliminated low moi spread of both mutant and WT rSV5 infections. Together, these data demonstrate that SV5 growth in normal primary human cells is highly sensitive to IFN compared to growth in some tumor cell lines, regardless of whether the P/V gene is WT or mutant. These results suggest a model in which spread of WT SV5 in normal human cells is dependent on the ability of the virus to prevent IFN synthesis. The

  11. Reasoning Abilities in Primary School: A Pilot Study on Poor Achievers vs. Normal Achievers in Computer Game Tasks

    ERIC Educational Resources Information Center

    Dagnino, Francesca Maria; Ballauri, Margherita; Benigno, Vincenza; Caponetto, Ilaria; Pesenti, Elia

    2013-01-01

    This paper presents the results of preliminary research on the assessment of reasoning abilities in primary school poor achievers vs. normal achievers using computer game tasks. Subjects were evaluated by means of cognitive assessment on logical abilities and academic skills. The aim of this study is to better understand the relationship between…

  12. Primary targets in photochemical inactivation of cells in culture

    NASA Astrophysics Data System (ADS)

    Berg, Kristian; Jones, Stuart G.; Prydz, Kristian; Moan, Johan

    1995-01-01

    The mechanisms of photoinactivation of NHIK 3025 cells in culture sensitized by tetrasulfonated phenylporphines (TPPS4) are described). Ultracentrifugation studies on postnuclear supernatants indicated that the intracellular distribution of TPPS4 resembles that of (beta) -N-acetyl-D-glucosaminidase ((beta) -AGA), a lysosomal marker enzyme, and that the cytosolic content of TPPS4 is below the detection limit of the ultracentrifugation method. Upon light exposure more than 90% of TPPS4 was lost from the lysosomal fractions, due to lysosomal rupture. The content of TPPS4 in the postnuclear supernatants was reduced by 30 - 40% upon exposure to light. This is most likely due to binding of TPPS4 to the nuclei, which were removed from the cell extracts before ultracentrifugation, after photochemical treatment. The unpolymerized form of tubulin seems to be an important target for the photochemical inactivation of NHIK 3025 cells. Since TPPS4 is mainly localized in lysosomes it was assumed that a dose of light disrupting a substantial number of lysosomes followed by microtubule depolymerization by nocodazole would enhance the sensitivity of the cells to photoinactivation. This was confirmed by using a colony-forming assay. The increased phototoxic effect exerted by such a treatment regime could be explained by an enhanced sensitivity of tubulin to light. Another cytosolic constituent, lactate dehydrogenase, was not photoinactivated by TPPS4 and light.

  13. Measurement tools and process indicators of patient safety culture in primary care. A mixed methods study by the LINNEAUS collaboration on patient safety in primary care

    PubMed Central

    Parker, Dianne; Wensing, Michel; Esmail, Aneez; Valderas, Jose M

    2015-01-01

    ABSTRACT Background: There is little guidance available to healthcare practitioners about what tools they might use to assess the patient safety culture. Objective: To identify useful tools for assessing patient safety culture in primary care organizations in Europe; to identify those aspects of performance that should be assessed when investigating the relationship between safety culture and performance in primary care. Methods: Two consensus-based studies were carried out, in which subject matter experts and primary healthcare professionals from several EU states rated (a) the applicability to their healthcare system of several existing safety culture assessment tools and (b) the appropriateness and usefulness of a range of potential indicators of a positive patient safety culture to primary care settings. The safety culture tools were field-tested in four countries to ascertain any challenges and issues arising when used in primary care. Results: The two existing tools that received the most favourable ratings were the Manchester patient safety framework (MaPsAF primary care version) and the Agency for healthcare research and quality survey (medical office version). Several potential safety culture process indicators were identified. The one that emerged as offering the best combination of appropriateness and usefulness related to the collection of data on adverse patient events. Conclusion: Two tools, one quantitative and one qualitative, were identified as applicable and useful in assessing patient safety culture in primary care settings in Europe. Safety culture indicators in primary care should focus on the processes rather than the outcomes of care. PMID:26339832

  14. Assessment of patient safety culture in primary care setting, Al-Mukala, Yemen.

    PubMed

    Webair, Hana H; Al-Assani, Salwa S; Al-Haddad, Reema H; Al-Shaeeb, Wafa H; Bin Selm, Manal A; Alyamani, Abdulla S

    2015-10-13

    Patient safety culture in primary care is the first step to achieve high quality health care. This study aims to provide a baseline assessment of patient safety culture in primary care settings in Al-Mukala, Yemen as a first published study from a least developed country. A survey was conducted in primary healthcare centres and units in Al-Mukala District, Yemen. A comprehensive sample from the available 16 centres was included. An Arabic version of the Medical Office Survey on Patient Safety Culture was distributed to all health workers (110). Participants were physicians, nurses and administrative staff. The response rate from the participating centres was 71 %. (N = 78). The percent positive responses of the items is equal to the percentage of participants who answered positively. Composite scores were calculated by averaging the percent positive response on the items within a dimension. Positive safety culture was defined as 60 % or more positive responses on items or dimensions. Patient safety culture was perceived to be generally positive with the exception of the dimensions of 'Communication openness', 'Work pressure and pace' and 'Patient care tracking/follow-up', as the percent positive response of these dimensions were 58, 57, and 52 % respectively. Overall, positive rating on quality and patient safety were low (49 and 46 % respectively). Although patient safety culture in Al-Mukala primary care setting is generally positive, patient safety and quality rating were fairly low. Implementation of a safety and quality management system in Al-Mukala primary care setting are paramount. Further research is needed to confirm the applicability of the Medical Office Survey on Patient Safety Culture (MOSPSC) for Al-Mukala primary care.

  15. Evaluation of PFOS-mediated neurotoxicity in rat primary neurons and astrocytes cultured separately or in co-culture.

    PubMed

    Li, Zhenwei; Liu, Qi; Liu, Chang; Li, Chunna; Li, Yachen; Li, Shuangyue; Liu, Xiaohui; Shao, Jing

    2017-02-01

    Perfluorooctane sulfonate (PFOS) is a potential neurotoxicant reported by epidemiological investigations and experimental studies, while the underlying mechanisms are still unclear. Astrocytes not only support for the construction of neurons, but also conduct neuronal functions through glutamate-glutamine cycle in astrocyte-neuron crosstalk. In the present study, the effect of PFOS exposure on rat primary hippocampal neurons or cortex astrocytes was evaluated. Then the role of the astrocytes in PFOS-induced toxic effect on neurons was explored with astrocyte-neuron co-culture system. Exposure of rat primary hippocampal neurons to PFOS has led to oxidation-antioxidation imbalance, increased apoptosis and abnormal autophagy. The adverse effect of PFOS on rat primary cortex astrocytes manifested in the form of altered extracellular glutamate and glutamine concentrations, decreased glutamine synthase activity, as well as decreased gene expression of glutamine synthase, glutamate transporters and glutamine transporters in the glutamate-glutamine cycle. Especially, the alleviation of PFOS-inhibited neurite outgrowth in neurons could be observed in astrocyte-neuron co-culture system, though the ability of astrocytes in fostering neurite outgrowth was affected by PFOS. These results indicated that both astrocytes and neurons might be the targets of PFOS-induced neurotoxicity, and astrocytes could protect against PFOS-inhibited neurite outgrowth in primary cultured neurons. Our research might render some information in explaining the mechanisms of PFOS-induced neurotoxicity.

  16. Basolateral Cl channels in primary airway epithelial cultures.

    PubMed

    Fischer, Horst; Illek, Beate; Finkbeiner, Walter E; Widdicombe, Jonathan H

    2007-06-01

    Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance (G(Cl)) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral G(Cl) was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl(2) = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl(2) > tamoxifen > 4,4'-dinitro-2,2'-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of G(Cl) were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive G(Cl); 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive G(Cl); and 3) a forskolin-activated, linear G(Cl), which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl(-) secretion or support transcellular Cl(-) absorption.

  17. Conditions for initiating Lake Victoria haplochromine (Oreochromis esculentus) primary cell cultures from caudal fin biopsies.

    PubMed

    Filice, Melissa; Lee, C; Mastromonaco, Gabriela F

    2014-10-01

    The global decline of freshwater fishes has created a need to cryopreserve biological materials from endangered species in an effort to conserve the biodiversity within this taxon. Since maternal gametes and embryos from fish are difficult to cryopreserve, somatic cells obtained from caudal fins have become an increasingly popular resource as they contain both maternal and paternal DNA ensuring valuable traits are not lost from the population. Somatic cells stored in cryobanks can be used to supplement endangered populations with genetically valuable offspring with the use of assisted reproductive technologies. However, initiating primary cell cultures from caudal fin biopsies of endangered species can be challenging as standardized protocols have not yet been developed. The objective of this study was to identify culture conditions, including antibiotic supplementation, biopsy size, and culture temperature, suitable for establishing primary cell cultures of ngege (Oreochromis esculentus), a critically endangered African cichlid. Six-millimeter caudal fin biopsies provided sufficient material to develop a primary cell culture when incubated at 25°C using standard fish cell culture medium containing 1× Primocin. Further investigation and application of these culture conditions for other endangered freshwater fishes is necessary.

  18. [Primary culture and morphologic observation of eutopic and ectopic endometrial cells from patients with endometriosis].

    PubMed

    Yu, Chao-Qin; Shi, Shu-Fang; Liu, Yu-Huan; Wang, Rui-Xia; Song, Yan-Hua; Yu, Jin

    2006-03-01

    To explore the method of primary culture for endometriotic cells and to find out the differences in morphological manifestations among endometriotic cells and eutopic endometrial cells sampled from patients with endometriosis and endometriosis-free women. Endometriotic and eutopic endometrial cells were cultured by modified method of primary culture. The endometriotic cell types were observed and differentiated under optical and electron microscopes. The success rates for culture of eutopic endometrial cells from endometriosis-free women and patients with endometriosis were 91.67% and 93.75% respectively. The success rate for culture of endometriotic cells was 75.00%. The size of endometriotic glandular cells was similar to those of eutopic endometrial glandular cells from endometriosis-free women and patients with endometriosis. The chromatin was manifold and the nucleus was augmented in the endometriotic glandular cells. The endometriotic stromal cells were smaller than the eutopic endometrial stromal cells from endometriosis-free women and patients with endometriosis. Many tiny villi and protuberances on plasma membrane could be seen in the endometriotic stromal cells. The success rate for culture of endometriotic cells can be elevated through improving the method of primary culture. The ultrastructures of endometriotic glandular and stromal cells are obviously different from those of eutopic endometrial glandular and stromal cells from endometriosis-free women and patients with endometriosis.

  19. Patient safety culture in primary care: developing a theoretical framework for practical use

    PubMed Central

    Kirk, Susan; Parker, Dianne; Claridge, Tanya; Esmail, Aneez; Marshall, Martin

    2007-01-01

    Objective Great importance has been attached to a culture of safe practice in healthcare organisations, but it has proved difficult to engage frontline staff with this complex concept. The present study aimed to develop and test a framework for making the concept of safety culture meaningful and accessible to managers and frontline staff, and facilitating discussion of ways to improve team/organisational safety culture. Setting Eight primary care trusts and a sample of their associated general practices in north west England. Methods In phase 1 a comprehensive review of the literature and a postal survey of experts helped identify the key dimensions of safety culture in primary care. Semistructured interviews with 30 clinicians and managers explored the application of these dimensions to an established theory of organisational maturity. In phase 2 the face validity and utility of the framework was assessed in 33 interviews and 14 focus groups. Results Nine dimensions were identified through which safety culture is expressed in primary care organisations. Organisational descriptions were developed for how these dimensions might be characterised at five levels of organisational maturity. The resulting framework conceptualises patient safety culture as multidimensional and dynamic, and seems to have a high level of face validity and utility within primary care. It aids clinicians' and managers' understanding of the concept of safety culture and promotes discussion within teams about their safety culture maturity. Conclusions The framework moves the agenda on from rhetoric about the importance of safety culture to a way of understanding why and how the shared values of staff working within a healthcare organisation may be operationalised to create a safe environment for patient care. PMID:17693682

  20. 3D co-culturing model of primary pancreatic islets and hepatocytes in hybrid spheroid to overcome pancreatic cell shortage.

    PubMed

    Jun, Yesl; Kang, Ah Ran; Lee, Jae Seo; Jeong, Gi Seok; Ju, Jongil; Lee, Dong Yun; Lee, Sang-Hoon

    2013-05-01

    Here, a spheroidal 3D co-culture model of primary (rat) pancreatic islets and hepatocytes with uniform size and shape was developed using hemispheric concave microwell arrays. We conducted morphological and functional analyses of hybrid spheroids versus mono-cultures of islets or hepatocytes (controls). For the establishment of a 3D hybrid model, a broad range of cell ratios - 1:1, 1:3, 1:5, 1:7, 3:1, 5:1 and 7:1 mixture - of hepatocytes and pancreatic islets were used. As control, each hepatocyte and pancreatic islet were mono-cultured forming 3D spheroids. The transient morphology of spheroid formation in 9 culture models was observed using optical microscopy. Cell viability under these culture environments was assessed, and the morphologies of the outer and inner porous cell-spheroid structures were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and imaging of stained spheroid sections. The pancreatic islet-specific function of hybrid spheroids was evaluated by measuring insulin secretion and in vivo test by xenotransplantation of encapsulated spheroids in microfibers with a consistent maintenance of normal blood glucose levels over 4 weeks, while liver-specific functions were measured in terms of albumin secretion, urea secretion and cytochrome P450 activity. These diverse observations and evaluations validated the positive and bidirectional effects of co-cultured 3D spheroids. The proposed 3D co-culture model demonstrated that both cells appeared to support each other's functions strongly in spheroids, even though smaller proportions of each cell type was evaluated compared to mono-culture models, suggesting that the proposed model could help overcome the problem of cell shortages in clinical applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Hydroxylation, conjugation and sulfation of bile acids in primary monolayer cultures of rat hepatocytes

    SciTech Connect

    Princen, H.M.; Meijer, P.

    1988-08-15

    Hydroxylation of lithocholic, chenodeoxycholic, deoxycholic and cholic acids was studied in monolayers of rat hepatocytes cultured for 76 h. The majority of added lithocholic and chenodeoxycholic acids was metabolized to beta-muricholic acid (56-76%). A small part of these bile acids (9%), however, and a considerable amount of deoxycholic and cholic acids (21%) were converted into metabolites more polar than cholic acid in the first culture period. Formation of these compounds decreased during the last day of culture. Bile acids synthesized after addition of (4-/sup 14/C)-cholesterol were almost entirely (97%) sulfated and/or conjugated, predominantly with taurine (54-66%), during culture. Sulfated bile acids were mainly composed of free bile acids. The ability of hepatocytes to sulfurylate bile acids declined with culture age. Thus, rat hepatocytes in primary monolayer culture are capable to sulfurylate bile acids and to hydroxylate trihydroxylated bile acids, suggesting formation of polyhydroxylated metabolites.

  2. Organization Complexity and Primary Care Providers' Perceptions of Quality Improvement Culture Within the Veterans Health Administration.

    PubMed

    Korom-Djakovic, Danijela; Canamucio, Anne; Lempa, Michele; Yano, Elizabeth M; Long, Judith A

    2016-01-01

    This study examined how aspects of quality improvement (QI) culture changed during the introduction of the Veterans Health Administration (VHA) patient-centered medical home initiative and how they were influenced by existing organizational factors, including VHA facility complexity and practice location. A voluntary survey, measuring primary care providers' (PCPs') perspectives on QI culture at their primary care clinics, was administered in 2010 and 2012. Participants were 320 PCPs from hospital- and community-based primary care practices in Pennsylvania, West Virginia, Delaware, New Jersey, New York, and Ohio. PCPs in community-based outpatient clinics reported an improvement in established processes for QI, and communication and cooperation from 2010 to 2012. However, their peers in hospital-based clinics did not report any significant improvements in QI culture. In both years, compared with high-complexity facilities, medium- and low-complexity facilities had better scores on the scales assessing established processes for QI, and communication and cooperation.

  3. Spectrum of Cytogenomic Abnormalities Revealed by Array Comparative Genomic Hybridization on Products of Conception Culture Failure and Normal Karyotype Samples.

    PubMed

    Zhou, Qinghua; Wu, Shen-Yin; Amato, Katherine; DiAdamo, Autumn; Li, Peining

    2016-03-20

    Approximately 30% of pregnancies after implantation end up in spontaneous abortions, and 50% of them are caused by chromosomal abnormalities. However, the spectrum of genomic copy number variants (CNVs) in products of conception (POC) and the underlying gene-dosage-sensitive mechanisms causing spontaneous abortions remain largely unknown. In this study, array comparative genomic hybridization (aCGH) analysis was performed as a salvage procedure for 128 POC culture failure (POC-CF) samples and as a supplemental procedure for 106 POC normal karyotype (POC-NK) samples. Chromosomal abnormalities were detected in 10% of POC-CF and pathogenic CNVs were detected in 3.9% of POC-CF and 5.7% of POC-NK samples. Compiled results from this study and relevant case series through a literature review demonstrated an abnormality detection rate (ADR) of 35% for chromosomal abnormalities in POC-CF samples, 3.7% for pathogenic CNVs in POC-CF samples, and 4.6% for pathogenic CNVs in POC-NK samples. Ingenuity Pathway Analysis (IPA) was performed on the genes from pathogenic CNVs found in POC samples. The denoted primary gene networks suggested that apoptosis and cell proliferation pathways are involved in miscarriage. In summary, a similar spectrum of cytogenomic abnormalities was observed in POC culture success and POC-CF samples. A threshold effect correlating the number of dosage-sensitive genes in a chromosome with the observed frequency of autosomal trisomy is proposed. A rationalized approach using firstly fluorescence in situ hybridization (FISH) testing with probes of chromosomes X/Y/18, 13/21, and 15/16/22 for common aneuploidies and polyploidies and secondly aCGH for other cytogenomic abnormalities is recommended for POC-CF samples.

  4. Control of thyrotropin glycosylation in normal rat pituitary cells in culture: effect of thyrotropin-releasing hormone

    SciTech Connect

    Ponsin, G.; Mornex, R.

    1983-08-01

    Regulation of glycosylation of TSH was studied in primary cultures of normal rat pituitary cells. (3H)Glucosamine or (3H)proline incorporation into immunoprecipitable TSH and trichloroacetic acid-precipitable proteins was measured after incubation periods ranging from 4-72 h. TSH release was assessed by RIA of TSH in the medium. TRH (30 nM) specifically increased the glycosylation of TSH despite the fact that it did not stimulate (3H)proline incorporation into the hormone even after 72 h of continuous labeling. The TRH-stimulated (3H)glucosamine-labeled TSH was completely recovered in the incubation medium. Effective concentrations of TRH were in the same range as those necessary for stimulation of TSH release (10(-10) - 10(-6) M). Somatostatin (50 nM) and T3 (10 microM) antagonized TRH effects on both TSH release and glycosylation. Stages of TSH glycosylation were discriminated by the addition to the culture medium of tunicamycin (10 micrograms/ml) or monensin (25 microM), which are known to inhibit core and terminal glycosylation of proteins, respectively. Medium (3H)glucosamine-labeled TSH was fully glycosylated, whereas a large part of the intracellular hormone was only core glycosylated. This suggests that terminal glycosylation of TSH could be related to hormone secretion. TRH stimulated essentially only terminal glycosylation of TSH. No alteration of core glycosylation of the hormone was observed after TRH treatment. The stimulating effect of TRH on terminal glycosylation of TSH is probably related to its ability to stimulate hormone release.

  5. Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

    PubMed Central

    Carney, Colleen M.; Muszynski, Jessica L.; Strotman, Lindsay N.; Lewis, Samantha R.; O'Connell, Rachel L.; Beebe, David J.; Theberge, Ashleigh B.; Jorgensen, Joan S.

    2014-01-01

    ABSTRACT Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3–5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture. PMID:25143354

  6. Cellular microenvironment dictates androgen production by murine fetal Leydig cells in primary culture.

    PubMed

    Carney, Colleen M; Muszynski, Jessica L; Strotman, Lindsay N; Lewis, Samantha R; O'Connell, Rachel L; Beebe, David J; Theberge, Ashleigh B; Jorgensen, Joan S

    2014-10-01

    Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3-5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture.

  7. Impact of Different Normality Thresholds for 24-hour ABPM at the Primary Health Care Level.

    PubMed

    Grezzana, Guilherme Brasil; Moraes, David William; Stein, Airton Tetelbon; Pellanda, Lucia Campos

    2017-02-01

    Hypertension is an important risk factor for cardiovascular outcomes. Primary health care (PHC) physicians should be prepared to act appropriately in the prevention of cardiovascular risk factors. However, the rates of patients with control of blood pressure (BP) remain low. The impact of the reclassification of high BP by 24-hour ambulatory BP monitoring (ABPM) can lead to different medical decisions in PHC. To evaluate the agreement between the BP measured by a conventional method by PHC physicians and by 24-hour ABPM, considering different BP normal thresholds for the 24-hour ABPM according to the V Brazilian ABPM Guidelines and the European Society of Hypertension Guidelines. A cross-sectional study including 569 hypertensive patients. The BP was initially measured by the PHC physicians and, later, by 24-hour ABPM. The BP measurements were obtained independently between the two methods. The therapeutic targets for the conventional BP followed the guidelines by the Eighth Joint National Committee (JNC 8), the V ABPM Brazilian Guidelines, and the 2013 European Hypertension Guidelines. There was an accuracy of 54.8% (95% confidence interval [95%CI] 0.51 - 0.58%) for the BP measured with the conventional method when compared with the 24-hour ABPM, with a sensitivity of 85% (95%CI 80.8 - 88.6%), specificity of 31.9% (95%CI 28.7 - 34.7%), and kappa value of 0.155, when considering the European Hypertension Guidelines. When using more stringent thresholds to characterize the BP as "normal" by ABPM, the accuracy was 45% (95%CI 0.41 - 0.47%) for conventional measurement when compared with 24-hour ABPM, with a sensitivity of 86.7% (95%CI 0.81 - 0.91%), specificity of 29% (95%CI 0.26 - 0.30%), and kappa value of 0.103. The BP measurements obtained by PHC physicians showed low accuracy when compared with those obtained by 24-hour ABPM, regardless of the threshold set by the different guidelines. A hipertensão arterial sistêmica é um fator de risco importante para

  8. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures

    PubMed Central

    Lepsch, Lucilia B.; Planeta, Cleopatra S.; Scavone, Critoforo

    2015-01-01

    To study cocaine's toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2) and/or neuronal nucleus protein (NeuN) staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine. PMID:26295051

  9. Primary culture of strial marginal cells of guinea pig cochlea: growth, morphologic features, and characterization.

    PubMed

    Achouche, J; Liu, D S; Tran Ba Huy, P; Huy, P T

    1991-12-01

    To further investigate the cellular mechanisms involved in the formation of endolymph, primary cultures of marginal cells of guinea pig were established. Minute explants obtained by mechanical dissociation of stria vascularis were plated on collagen type I precoated impermeable substrate in serum-free, hormone-supplemented medium. A confluent layer of epithelial-like cells was obtained within 2 weeks. The cultured cells formed domes, demonstrating that they retain some of their transepithelial properties. Polarization was also suggested by electron microscopic observation of apical microvilli and tight junctions. Immunohistochemical methods revealed that the cultured cells coexpressed cytokeratin and vimentin, demonstrating their epithelial origin, although some degree of dedifferentiation occurred. Thus, a primary culture of marginal cells can be established that may be a suitable model for an in-depth investigation of the function of the marginal cells.

  10. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures.

    PubMed

    Lepsch, Lucilia B; Planeta, Cleopatra S; Scavone, Critoforo

    2015-01-01

    To study cocaine's toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2) and/or neuronal nucleus protein (NeuN) staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine.

  11. The proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°Celsius compared to euthermic conditions in pigs.

    PubMed

    Bohan, Amy E; Purvis, Katelyn N; Bartosh, Julia L; Brandebourg, Terry D

    2014-01-01

    Given similarities in metabolic parameters and cardiovascular physiology, the pig is well positioned as a biomedical model for metabolic disease and obesity in humans. Better understanding molecular mechanisms governing porcine adipocyte hyperplasia may provide insight into the regulation of adipose tissue development that is useful both when considering the pig as a commodity and when extrapolating porcine data to human disease. Primary cultures of pig stromal-vascular cells have served as a useful tool for investigating factors that regulate preadipocyte proliferation and differentiation. However, such cultures have generally been maintained at 37°C in vitro despite euthermia being 39°C in pigs. To address potential concerns about the physiological relevance of culturing primary pig preadipocytes under what would be hypothermic conditions in vivo, the objective of this study was to investigate the effect of culture temperature on the proliferation and differentiation of pig preadipocytes in primary culture. Culturing primary preadipocytes at 37 rather than 39°C decreases their proliferation rates based upon cleavage of the tetrazolium salt, MTT (P < 0.001), reduction of resazurin (P < 0.001), and daily cell counts (P < 0.001). Likewise, culturing primary porcine preadipocytes at 37°C suppressed their adipogenic potential based upon monitoring adipogenesis morphologically, biochemically, and via the expression of mRNA encoding adipogenic marker genes. Collectively, these data indicate the proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°C compared to normal body temperature of pigs. This may confound investigation of factors that impact adipocyte hyperplasia in the pig.

  12. Optimization of NRU assay in primary cultures of Eisenia fetida for metal toxicity assessment.

    PubMed

    Irizar, Amaia; Duarte, Daniel; Guilhermino, Lucia; Marigómez, Ionan; Soto, Manu

    2014-09-01

    Coelomocytes, immunocompetent cells of lumbricids, have received special attention for ecotoxicological studies due to their sensibility to pollutants. Their in vitro responses are commonly quantified after in vivo exposure to real or spiked soils. Alternatively, quantifications of in vitro responses after in vitro exposure are being studied. Within this framework, the present study aimed at optimizing the neutral red uptake (NRU) assay in primary culture of Eisenia fetida coelomocytes for its application in soil toxicity testing. Optimized assay conditions were: earthworm depuration for 24 h before retrieving coelomocytes by electric extrusion; 2 × 10(5) seeded cells/well (200 µl) for the NRU assay and incubation for 1 h with neutral red dye. Supplementation of the culture medium with serum was not compatible with the NRU assay, but coelomocytes could be maintained with high viability for 3 days in a serum-free medium without replenishment. Thus, primary cultures were used for 24 h in vitro toxicity testing after exposure to different concentrations of Cd, Cu, Ni and Pb (ranging from 0.1 to 100 μg/ml). Primary cultures were sensitive to metals, the viability declining in a dose-dependent manner. The toxicity rank was, from high to low, Pb > Ni > Cd > Cu. Therefore, it can be concluded that the NRU assay in coelomocytes in primary cultures provides a sensitive and prompt response after in vitro exposure to metals.

  13. Methods Development for the Isolation and Culture of Primary Corneal Endothelial Cells

    DTIC Science & Technology

    2017-02-01

    USAMRICD‐TR‐17‐02  Methods Development for the Isolation and  Culture of Primary Corneal Endothelial  Cells       Robert L. Haggins Jr...Development for the Isolation and Culture of Primary Corneal Endothelial Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6...endothelial cells (CECs). If so, cellular therapy intended to replenish CECs lost to injury and age may represent an effective approach in the

  14. Health system factors affecting communication with pediatricians: gendered work culture in primary care.

    PubMed

    Lynch, Sean

    2011-01-01

    This qualitative study examined the roles that practice setting, education level, and gender may play in social workers' communication satisfaction with pediatricians. Taking an ethnographic approach, the researcher interviewed social workers and pediatricians who worked together to provide mental health services in primary care. The results suggested that gender at the health system level may be an issue and that gendered work culture in primary care was a factor in communication. In particular, reimbursement, an aspect of the gendered work culture, was a substantial communication barrier, and the implications for Medicaid billing are discussed.

  15. Normal human serum (HS) prevents oxidant-induced lysis of cultured endothelial cells (ECs)

    SciTech Connect

    Callahan, K.S.; Harlan, J.M.

    1986-03-01

    Most studies demonstrating oxidant lysis of cultured ECs are performed in serum-free media or media containing low concentrations of bovine serum. The authors found that HS protects human and bovine ECs from lysis caused by reagent H/sub 2/O/sub 2/ or glucose/glucose oxidase (GO)-generated H/sub 2/O/sub 2/. EC injury was assessed by /sup 51/Cr release, cell detachment, or trypan blue dye exclusion. Protective HS activity was dose-dependent with concentrations greater than or equal to 25% preventing lethal injury. Cytotoxicity at 24 hrs, induced by 20 mU/ml GO, was 90.1 +/- 5.2% without HS vs 1.7 +/- 4.6% with 25% HS present (20 exp). Similar protection was observed with heparinized plasma. Of note, comparable concentrations of bovine serum were devoid of protective activity. Addition of fatty acid-free albumin to the media was also without protective effect. Preliminary characterization showed HS activity was stable to 60/sup 0/C for 30 min, non-dialyzable at 25,000 MW cutoff, and retained in delipidated serum. The HS protection was not merely due to scavenging of exogenous H/sub 2/O/sub 2/ as A23187-induced EC lysis was also prevented by HS. Protective activity was not reproduced by purified cerruloplasmin or transferrin. In conclusion, unidentified factor(s) present in HS protect cultured ECs from oxidant-induced lysis. Since endothelium is normally exposed to 100% plasma, the authors suggest that in vitro studies of oxidant-mediated injury be performed in the presence of HS. Factor(s) in HS may play an important role in modulating oxidant-induced vascular injury in vivo.

  16. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    SciTech Connect

    Novicki, D.L.; Rosenberg, M.R.; Michalopoulos, G.

    1985-01-01

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of (3H)thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity.

  17. Establishment of primary cell culture from the temperate symbiotic cnidarian, Anemonia viridis.

    PubMed

    Barnay-Verdier, Stéphanie; Dall'osso, Diane; Joli, Nathalie; Olivré, Juliette; Priouzeau, Fabrice; Zamoum, Thamilla; Merle, Pierre-Laurent; Furla, Paola

    2013-10-01

    The temperate symbiotic sea anemone Anemonia viridis, a member of the Cnidaria phylum, is a relevant experimental model to investigate the molecular and cellular events involved in the preservation or in the rupture of the symbiosis between the animal cells and their symbiotic microalgae, commonly named zooxanthellae. In order to increase research tools for this model, we developed a primary culture from A. viridis animal cells. By adapting enzymatic dissociation protocols, we isolated animal host cells from a whole tentacle in regeneration state. Each plating resulted in a heterogeneous primary culture consisted of free zooxanthellae and many regular, small rounded and adherent cells (of 3-5 μm diameter). Molecular analyses conducted on primary cultures, maintained for 2 weeks, confirmed a specific signature of A. viridis cells. Further serial dilutions and micromanipulation allowed us to obtain homogenous primary cultures of the small rounded cells, corresponding to A. viridis "epithelial-like cells". The maintenance and the propagation over a 4 weeks period of primary cells provide, for in vitro cnidarian studies, a preliminary step for further investigations on cnidarian cellular pathways notably in regard to symbiosis interactions.

  18. Morphology of primary human venous endothelial cell cultures before and after culture medium exchange.

    PubMed

    Krüger-Genge, A; Fuhrmann, R; Jung, F; Franke, R P

    2015-01-01

    The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo, but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function was investigated. HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS - Image analysis system. Prostacyclin was analysed in the cell culture supernatant. 1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the

  19. Preventive effect of piracetam and vinpocetine on hypoxia-reoxygenation induced injury in primary hippocampal culture.

    PubMed

    Solanki, P; Prasad, D; Muthuraju, S; Sharma, A K; Singh, S B; Ilavzhagan, G

    2011-04-01

    The present study investigates the potential of Piracetam and Vinpocetine (nootropic drugs, known to possess neuroprotective properties) in preventing hypoxia-reoxygenation induced oxidative stress in primary hippocampal cell culture. The hippocampal culture was exposed to hypoxia (95% N(2), 5% CO(2)) for 3h and followed by 1h of reoxygenation (21% O(2) and 5% CO(2)) at 37 °C. The primary hippocampal cultures were supplemented with the optimum dose of Piracetam and Vinpocetine, independently, and the cultures were divided into six groups, viz. Control/Normoxia, Hypoxia, Hypoxia+Piracetam, Hypoxia+Vinpocetine, Normoxia + Piracetam and Normoxia+Vinpocetine. The cell-viability assays and biochemical oxidative stress parameters were evaluated for each of the six groups. Administration of 1mM Piracetam or 500 nM Vinpocetine significantly prevents the culture from hypoxia-reoxygenation injury when determined by Neutral Red assay, LDH release and Acetylcholine esterase activity. Results showed that Piracetam and Vinpocetine supplementation significantly prevented the fall of mitochondrial membrane potential, rise in ROS generation and reduction in antioxidant levels associated with the hypoxia-reoxygenation injury. In conclusion, the present study establishes that both Piracetam and Vinpocetine give neuroprotection against hypoxia-reoxygenation injury in primary hippocampal cell culture. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids.

    PubMed

    DiMarco, Rebecca L; Dewi, Ruby E; Bernal, Gabriela; Kuo, Calvin; Heilshorn, Sarah C

    2015-10-15

    Though in vitro culture of primary intestinal organoids has gained significant momentum in recent years, little has been done to investigate the impact of microenvironmental cues provided by the encapsulating matrix on the growth and development of these fragile cultures. In this work, the impact of various in vitro culture parameters on primary adult murine organoid formation and growth are analyzed with a focus on matrix properties and geometric culture configuration. The air-liquid interface culture configuration was found to result in enhanced organoid formation relative to a traditional submerged configuration. Additionally, through use of a recombinantly engineered extracellular matrix (eECM), the effects of biochemical and biomechanical cues were independently studied. Decreasing mechanical stiffness and increasing cell adhesivity were found to increase organoid yield. Tuning of eECM properties was used to obtain organoid formation efficiency values identical to those observed in naturally harvested collagen I matrices but within a stiffer construct with improved ease of physical manipulation. Increased ability to remodel the surrounding matrix through mechanical or enzymatic means was also shown to enhance organoid formation. As the engineering and tunability of recombinant matrices is essentially limitless, continued property optimization may result in further improved matrix performance and may help to identify additional microenvironmental cues that directly impact organoid formation, development, differentiation, and functional behavior. Continued culture of primary organoids in recombinant matrices could therefore prove to be largely advantageous in the field of intestinal tissue engineering for applications in regenerative medicine and in vitro tissue mimics.

  1. Efficient and simple approach to in vitro culture of primary epithelial cancer cells

    PubMed Central

    Janik, Karolina; Popeda, Marta; Peciak, Joanna; Rosiak, Kamila; Smolarz, Maciej; Treda, Cezary; Rieske, Piotr; Stoczynska-Fidelus, Ewelina; Ksiazkiewicz, Magdalena

    2016-01-01

    Primary cancer cells constitute a favourable testing platform for in vitro research in oncology field as they reflect tumour state more accurately than the most commonly employed stable cell lines. Unfortunately, due to limited availability of material and difficulties with protocols validation, primary models are rarely implemented into laboratory practice. We have compared protocols for primary cultures, differing in media components and plate coatings. In terms of culture establishment, application of Geltrex® coating demonstrated equal efficiency to feeder layer (83% compared with 72% successfully established breast and 80% compared with 80% prostate tumour specimens), yet it was substantially less complicated and easier to validate. Both Geltrex® coating and tissue-specific primary cell medium were permanently required to successfully maintain primary epithelial prostate cancer cells (PEPCs) in culture. In case of primary epithelial breast cancer cells (PEBCs), collagen I coating enabled to obtain comparable number of passages to Geltrex® coating (P=0.438). Commercial primary cell media demonstrated lower efficiency than tissue-specific ones (PEPCs–5 compared with 8 and PEBCs–6 compared with 9 passages). Interestingly, both analysed tumour types were unsusceptible to induction of culture lifespan extension when transduced with SV40LT, BMI-1 or hEST2 genes, commonly applied as potential immortalizing agents. In conclusion, the approach based on extracellular matrix reconstitution and tissue-specific primary cell media is easy to validate and provides in vitro expansion sufficient for analytical purposes (approximately 8 passages). Therefore, it may facilitate implementation of hardly available experimental models for a variety of analyses. PMID:27803125

  2. Evidence for multiple bone resorption-stimulating factors produced by normal human keratinocytes in culture.

    PubMed

    Fried, R M; Voelkel, E F; Rice, R H; Levine, L; Tashjian, A H

    1988-06-01

    Conditioned medium from cultured normal human foreskin keratinocytes enhanced the release of calcium from neonatal mouse calvaria in organ culture. Unfractionated keratinocyte-conditioned medium (KCM) stimulated bone resorption in a dose-dependent manner, but it did not increase the concentration of prostaglandin E2 (PGE2) in the bone culture medium until a maximal dose of KCM for resorption was used. Furthermore, inhibitors of PGE2 synthesis, indomethacin, ibuprofen, and piroxicam, did not inhibit KCM-induced calcium release. High concentrations of KCM increased cAMP production by calvaria in the presence of isobutylmethylxanthine, but the increase was small compared with that produced by a dose of bovine PTH that caused a similar level of bone resorption. The bone resorption-stimulating activity of KCM was not lost after incubation at 56 C for 60 min, but it was lost after heating at 100 C for 10 min. Fractionation of KCM by gel filtration chromatography revealed two distinct peaks of bone resorption-stimulating activity. One peak, KCMI, caused a significant increase in bone resorption at 2 micrograms protein/ml. KCMI did not increase medium PGE2, and inhibition of PGE2 synthesis in bone had no effect on KCMI-induced bone resorption. KCMI failed to increase cAMP production by human osteosarcoma SaOS-2 cells. Another peak, KCMII, caused a dose-dependent increase in bone resorption, and a significant increase in medium calcium was noted at a 20-fold lower concentration (0.1 microgram protein/ml) than with KCMI. In contrast to KCMI, the increase in bone resorption stimulated by KCMII was accompanied by a parallel increase in the production of PGE2, and inhibition of PGE2 synthesis completely inhibited the bone resorption-stimulating activity of KCMII. KCMII also caused an increase in cAMP production by SaOS-2 cells. We conclude that KCM contains at least two distinct bone resorption-stimulating factors, one of which acts via a PG-mediated mechanism and the other by

  3. Differential susceptibility of primary cultured human skin cells to hypericin PDT in an in vitro model.

    PubMed

    Popovic, A; Wiggins, T; Davids, L M

    2015-08-01

    Skin cancer is the most common cancer worldwide, and its incidence rate in South Africa is increasing. Photodynamic therapy (PDT) has been shown to be an effective treatment modality, through topical administration, for treatment of non-melanoma skin cancers. Our group investigates hypericin-induced PDT (HYP-PDT) for the treatment of both non-melanoma and melanoma skin cancers. However, a prerequisite for effective cancer treatments is efficient and selective targeting of the tumoral cells with minimal collateral damage to the surrounding normal cells, as it is well established that cancer therapies have bystander effects on normal cells in the body, often causing undesirable side effects. The aim of this study was to investigate the cellular and molecular effects of HYP-PDT on normal primary human keratinocytes (Kc), melanocytes (Mc) and fibroblasts (Fb) in an in vitro tissue culture model which represented both the epidermal and dermal cellular compartments of human skin. Cell viability analysis revealed a differential cytotoxic response to a range of HYP-PDT doses in all the human skin cell types, showing that Fb (LD50=1.75μM) were the most susceptible to HYP-PDT, followed by Mc (LD50=3.5μM) and Kc (LD50>4μM HYP-PDT) These results correlated with the morphological analysis which displayed distinct morphological changes in Fb and Mc, 24h post treatment with non-lethal (1μM) and lethal (3μM) doses of HYP-PDT, but the highest HYP-PDT doses had no effect on Kc morphology. Fluorescent microscopy displayed cytoplasmic localization of HYP in all the 3 skin cell types and additionally, HYP was excluded from the nuclei in all the cell types. Intracellular ROS levels measured in Fb at 3μM HYP-PDT, displayed a significant 3.8 fold (p<0.05) increase in ROS, but no significant difference in ROS levels occurred in Mc or Kc. Furthermore, 64% (p<0.005) early apoptotic Fb and 20% (p<0.05) early apoptotic Mc were evident; using fluorescence activated cell sorting (FACS), 24

  4. Evaluation of directional normalization methods for Landsat TM/ETM+ over primary Amazonian lowland forests

    NASA Astrophysics Data System (ADS)

    Van doninck, Jasper; Tuomisto, Hanna

    2017-06-01

    Biodiversity mapping in extensive tropical forest areas poses a major challenge for the interpretation of Landsat images, because floristically clearly distinct forest types may show little difference in reflectance. In such cases, the effects of the bidirectional reflection distribution function (BRDF) can be sufficiently strong to cause erroneous image interpretation and classification. Since the opening of the Landsat archive in 2008, several BRDF normalization methods for Landsat have been developed. The simplest of these consist of an empirical view angle normalization, whereas more complex approaches apply the semi-empirical Ross-Li BRDF model and the MODIS MCD43-series of products to normalize directional Landsat reflectance to standard view and solar angles. Here we quantify the effect of surface anisotropy on Landsat TM/ETM+ images over old-growth Amazonian forests, and evaluate five angular normalization approaches. Even for the narrow swath of the Landsat sensors, we observed directional effects in all spectral bands. Those normalization methods that are based on removing the surface reflectance gradient as observed in each image were adequate to normalize TM/ETM+ imagery to nadir viewing, but were less suitable for multitemporal analysis when the solar vector varied strongly among images. Approaches based on the MODIS BRDF model parameters successfully reduced directional effects in the visible bands, but removed only half of the systematic errors in the infrared bands. The best results were obtained when the semi-empirical BRDF model was calibrated using pairs of Landsat observation. This method produces a single set of BRDF parameters, which can then be used to operationally normalize Landsat TM/ETM+ imagery over Amazonian forests to nadir viewing and a standard solar configuration.

  5. Ultraviolet radiation acts as an independent mitogen for normal human melanocytes in culture

    SciTech Connect

    Libow, L.F.; Scheide, S.; DeLeo, V.A.

    1988-01-01

    Identification of growth factors for normal human melanocytes has been significantly aided by the recent development of in vitro culture systems for this cell. Utilizing such a system, we studied the effect of ultraviolet radiation (UVR) on both melanocyte growth and melanization by incorporation of 3H-thymidine and 3H-L-dihydroxyphenylalanine (3H-DOPA), respectively. 3H-thymidine incorporation was found to be significantly stimulated during the first 24 h following a single irradiation. 3H-DOPA incorporation was stimulated after a delay of 2 days postirradiation. Whereas UVR has long been known to induce melanocyte proliferation in vivo, these studies show that UVR can act as a mitogenic stimulus for this cell independent of the cutaneous environment. UVR can thus be added to a growing list of growth factors for epidermal pigment cells and is the only physical agent conclusively shown to act as a mitogen. Included in this list are substances that act via stimulation of the CAMP-kinase or protein kinase systems such as cholera toxin and phorbol esters. UVR is postulated to induce melanocyte proliferation by modulation of these second messenger pathways. With recent evidence linking growth factors, oncogenes and malignant transformation, this study supports the association between UVR exposure and the development of malignant melanoma, and suggests mechanisms whereby UVR may contribute to malignant transformation of this cell.

  6. Establishment of primary mixed cell cultures from spontaneous canine mammary tumors: Characterization of classic and new cancer-associated molecules

    PubMed Central

    Gentile, Luciana B.; Nagamine, Marcia K.; Biondi, Luiz R.; Sanches, Daniel S.; Toyota, Fábio; Giovani, Tatiane M.; de Jesus, Isis P.; da Fonseca, Ivone I. M.; Queiroz-Hazarbassanov, Nicolle; Diaz, Bruno L.; Salles Gomes, Cristina de O. Massoco

    2017-01-01

    There are many factors which make canine cancer like cancer in humans. The occurrence of spontaneous mammary tumors in pet dogs, tumor genetics, molecular targets and exposure to the same environmental risk factors are among these factors. Therefore, the study of canine cancer can provide useful information to the oncology field. This study aimed to establish and characterize a panel of primary mixed cell cultures obtained from spontaneous canine mammary tumors. Eight established cell cultures obtained from one normal mammary gland, one complex adenoma, one mixed adenoma, two complex carcinomas and two mixed carcinomas were analyzed. The gene expression levels of classic molecular cancer players such as fibroblast growth factor receptor (FGFR) 2, breast cancer (BRCA) 1, BRCA2 and estrogen receptor (ESR) 1 were evaluated. For the first time, three orphan nuclear receptors, estrogen-related receptors (ERRs) α, β and γ were studied in canine mammary cancer. The highest expression level of ERRα was observed in complex carcinoma-derived cell culture, while the highest levels of ERRβ and γ were observed in cells derived from a mixed carcinoma. Meanwhile, complex carcinomas presented the highest levels of expression of ESR1, BRCA1 and FGFR2 among all samples. BRCA2 was found exclusively in complex adenoma. The transcription factor GATA3 had its highest levels in mixed carcinoma samples and its lowest levels in complex adenoma. Proliferation assays were also performed to evaluate the mixed cell cultures response to ER ligands, genistein and DES, both in normoxia and hypoxic conditions. Our results demonstrate that morphological and functional studies of primary mixed cell cultures derived from spontaneous canine mammary tumors are possible and provide valuable tool for the study of various stages of mammary cancer development. PMID:28945747

  7. Establishment of primary mixed cell cultures from spontaneous canine mammary tumors: Characterization of classic and new cancer-associated molecules.

    PubMed

    Gentile, Luciana B; Nagamine, Marcia K; Biondi, Luiz R; Sanches, Daniel S; Toyota, Fábio; Giovani, Tatiane M; de Jesus, Isis P; da Fonseca, Ivone I M; Queiroz-Hazarbassanov, Nicolle; Diaz, Bruno L; Salles Gomes, Cristina de O Massoco; Dagli, Maria Lucia Z

    2017-01-01

    There are many factors which make canine cancer like cancer in humans. The occurrence of spontaneous mammary tumors in pet dogs, tumor genetics, molecular targets and exposure to the same environmental risk factors are among these factors. Therefore, the study of canine cancer can provide useful information to the oncology field. This study aimed to establish and characterize a panel of primary mixed cell cultures obtained from spontaneous canine mammary tumors. Eight established cell cultures obtained from one normal mammary gland, one complex adenoma, one mixed adenoma, two complex carcinomas and two mixed carcinomas were analyzed. The gene expression levels of classic molecular cancer players such as fibroblast growth factor receptor (FGFR) 2, breast cancer (BRCA) 1, BRCA2 and estrogen receptor (ESR) 1 were evaluated. For the first time, three orphan nuclear receptors, estrogen-related receptors (ERRs) α, β and γ were studied in canine mammary cancer. The highest expression level of ERRα was observed in complex carcinoma-derived cell culture, while the highest levels of ERRβ and γ were observed in cells derived from a mixed carcinoma. Meanwhile, complex carcinomas presented the highest levels of expression of ESR1, BRCA1 and FGFR2 among all samples. BRCA2 was found exclusively in complex adenoma. The transcription factor GATA3 had its highest levels in mixed carcinoma samples and its lowest levels in complex adenoma. Proliferation assays were also performed to evaluate the mixed cell cultures response to ER ligands, genistein and DES, both in normoxia and hypoxic conditions. Our results demonstrate that morphological and functional studies of primary mixed cell cultures derived from spontaneous canine mammary tumors are possible and provide valuable tool for the study of various stages of mammary cancer development.

  8. Thyrotropin Stimulates Differentiation Not Proliferation of Normal Human Thyrocytes in Culture

    PubMed Central

    Morgan, Sarah J.; Neumann, Susanne; Marcus-Samuels, Bernice; Gershengorn, Marvin C.

    2016-01-01

    Although TSH has been suggested to be a proliferative agent for thyrocytes, the effect of TSH on human thyroid cells remains controversial. In particular, most of the reported studies relied primarily on changes in DNA synthesis but have not included measurement of the number of cells. We argue that only a direct count of cell number, demonstrating classical exponential expansion, serves as a valid measurement of proliferation. Thus, although some data support TSH as a proliferative agent, most do not provide conclusive evidence. To generate conclusive evidence with regard to a proliferative effect of TSH in human thyrocytes, we performed various experiments using primary cultures of human thyrocytes. In contrast to previous reports, TSH [±insulin-like growth factor 1 (IGF-1)] did not induce proliferation of thyrocytes under a variety of different conditions. However, TSH/IGF-1 cotreatment did upregulate thyroid-specific gene expression including thyroglobulin (TG) and TSHR in a manner consistent with cellular differentiation. Evidence for a proliferative effect of TSH has been used to inform the American Thyroid Association’s guidelines for the management of thyroid cancer patients, which include TSH suppression. While these recommendations are admittedly based on low- to moderate-quality evidence, TSH suppression is still widely used. We present data that question the consensus view that TSH promotes proliferation of human thyrocytes (upon which the American Thyroid Association’s guidelines are based) and suggest that additional studies, including randomized controlled trials, are warranted to address this important clinical question. PMID:28082948

  9. Cabergoline protects dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture.

    PubMed

    Meinel, J; Radad, K; Rausch, W-D; Reichmann, H; Gille, G

    2015-01-01

    In the present study, primary mesencephalic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effect of cabergoline, an ergoline D2 receptor agonist, against the pesticide and neurotoxin rotenone relevant to Parkinson disease (PD). Treatment of cultures with cabergoline alone significantly increased the number of tyrosine hydroxylase immunoreactive (THir) neurons and reduced the release of lactate dehydrogenase (LDH) into the culture medium compared to untreated controls. Against rotenone toxicity, cabergoline significantly rescued degenerating THir neurons, reduced the release of LDH into the culture medium and improved the morphology of surviving THir neurons. The neuroprotective effects afforded by cabergoline were independent of dopaminergic stimulation as blocking of dopamine receptors by the dopamine receptor antagonist sulpiride did not prevent them. Furthermore, rotenone-induced formation of reactive oxygen species (ROS) was significantly reduced by cabergoline. Although cabergoline increased the glutathione (GSH) content in the culture, the protective effect for dopaminergic neurons seemed not to be predominantly mediated by increasing GSH, as depletion of GSH by L-buthionine-(S,R)-sulfoximine (BSO), a GSH biosynthesis inhibitor, did not prevent cabergoline-mediated neuroprotection of THir neurons in rotenone-treated cultures. Moreover, cabergoline significantly increased the ATP/protein ratio in primary mesencephalic cell cultures when added alone or prior to rotenone treatment. These results indicate a neuroprotective effect of cabergoline for dopaminergic neurons against rotenone toxicity. This effect was independent of dopamine receptor stimulation and was at least partially mediated by reducing ROS production and increasing the ATP/protein ratio.

  10. [Application of the cultural competence model in the experience of care in nursing professionals Primary Care].

    PubMed

    Gil Estevan, María Dolores; Solano Ruíz, María Del Carmen

    2017-06-10

    To know the experiences and perceptions of nurses in providing care and health promotion, women belonging to groups at risk of social vulnerability, applying the model of cultural competence Purnell. Phenomenological qualitative study. Department of Health Elda. A total of 22 primary care professional volunteers. Semi-structured interviews and focus groups with recording and content analysis, according to the theory model of cultural competence. Socio-cultural factors influence the relationship between professionals and users of the system. The subtle racism and historical prejudices create uncomfortable situations and mistrust. The language barrier makes it difficult not only communication, but also the monitoring and control of the health-disease process. The physical appearance and stereotypes are determining factors for primary care professionals. Although perceived misuse of health services are also talking about changes. The spiritual aspects of religious beliefs alone are taken into account in the case of Muslim women, not being considered as important in the case of Gypsy women and Romanian women. To provide quality care, consistent and culturally competent, it is necessary to develop training programs for professionals in cultural competence, to know the culture of other, and work without preconceived ideas, and ethnocentric; since the greater the knowledge of the cultural group being served, the better the quality of care provided. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

  11. Use of primary cultures of Kenyon cells from bumblebee brains to assess pesticide side effects.

    PubMed

    Wilson, Daniel E; Velarde, Rodrigo A; Fahrbach, Susan E; Mommaerts, Veerle; Smagghe, Guy

    2013-09-01

    Bumblebees are important pollinators in natural and agricultural ecosystems. The latter results in the frequent exposure of bumblebees to pesticides. We report here on a new bioassay that uses primary cultures of neurons derived from adult bumblebee workers to evaluate possible side-effects of the neonicotinoid pesticide imidacloprid. Mushroom bodies (MBs) from the brains of bumblebee workers were dissected and dissociated to produce cultures of Kenyon cells (KCs). Cultured KCs typically extend branched, dendrite-like processes called neurites, with substantial growth evident 24-48 h after culture initiation. Exposure of cultured KCs obtained from newly eclosed adult workers to 2.5 parts per billion (ppb) imidacloprid, an environmentally relevant concentration of pesticide, did not have a detectable effect on neurite outgrowth. By contrast, in cultures prepared from newly eclosed adult bumblebees, inhibitory effects of imidacloprid were evident when the medium contained 25 ppb imidacloprid, and no growth was observed at 2,500 ppb. The KCs of older workers (13-day-old nurses and foragers) appeared to be more sensitive to imidacloprid than newly eclosed adults, as strong effects on KCs obtained from older nurses and foragers were also evident at 2.5 ppb imidacloprid. In conclusion, primary cultures using KCs of bumblebee worker brains offer a tool to assess sublethal effects of neurotoxic pesticides in vitro. Such studies also have the potential to contribute to the understanding of mechanisms of plasticity in the adult bumblebee brain.

  12. Antibiotic Supplements Affect Electrophysiological Properties and Excitability of Rat Hippocampal Pyramidal Neurons in Primary Culture

    PubMed Central

    Bahrami, Farideh; Janahmadi, Mahyar

    2013-01-01

    Introduction: Antibiotic supplements are regularly used in neuronal culture media to control contamination; however, they can interfere with the neuronal excitability and affect electrophysiological properties. Therefore, in this study, the effect of penicillin/streptomycin supplements on the spontaneous electrophysiological activity of hippocampal pyramidal neurons was examined. Methods: Electrophysiological whole-cell patch-clamp recordings from rat hippocampal pyramidal cells in primary culture were performed to investigate the effects of antibiotic supplements on the intrinsic excitability of cultured cells. Results: The present findings indicated that presence of antibiotic supplements (penicillin/streptomycin) in the culture medium altered the intrinsic electrical activity of hippocampal pyramidal neurons in primary culture. These alterations included: 1) depolarized resting membrane potential; 2) a significant enhancement in the after-hyperpolarization amplitude; 3) a significant increase in the area under the action potential and in the decay and rise time of the action potential; 4) a significant broadening of action potential and 5) a significant reduction in the firing frequency. Conclusion: These findings suggest that addition of antibiotic supplements to culture media influences the neuronal excitability and alters the electrophysiological properties of cultured neurons, possibly through changing the ionic conductance underlying neuronal excitability. PMID:23567852

  13. Microbiological and bioinformatics analysis of primary Sjögren’s syndrome patients with normal salivation§

    PubMed Central

    Siddiqui, Huma; Chen, Tsute; Aliko, Ardita; Mydel, Piotr M; Jonsson, Roland; Olsen, Ingar

    2016-01-01

    Background Reduced salivation is considered a major clinical feature of most but not all cases of primary Sjögren’s syndrome (pSS). Reduced saliva flow may lead to changes in the salivary microbiota. These changes have mainly been studied with culture that typically recovers only 65% of the bacteria present. Objective This study was to use high throughput sequencing, covering both cultivated and not-yet-cultivated bacteria, to assess the bacterial microbiota of whole saliva in pSS patients with normal salivation. Methods Bacteria of whole unstimulated saliva from nine pSS patients with normal salivation flow and from nine healthy controls were examined by high throughput sequencing of the hypervariable region V1V2 of 16S rRNA using the 454 GS Junior system. Raw sequence reads were subjected to a species-level, reference-based taxonomy assignment pipeline specially designed for studying the human oral microbial community. Each of the sequence reads was BLASTN-searched against a database consisting of reference sequences representing 1,156 oral and 12,013 non-oral species. Unassigned reads were then screened for high-quality non-chimeras and subjected to de novo species-level operational taxonomy unit (OTU) calling for potential novel species. Downstream analyses, including alpha and beta diversities, were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline. To reveal significant differences between the microbiota of control saliva and Sjögren’s saliva, a statistical method introduced in Metastats www.metastats.cbcb.umd.edu was used. Results Saliva of pSS patients with normal salivation had a significantly higher frequency of Firmicutes compared with controls (p=0.004). Two other major phyla, Synergistetes and Spirochaetes, were significantly depleted in pSS (p=0.001 for both). In addition, we saw a nearly 17% decrease in the number of genera in pSS (25 vs. 30). While Prevotella was almost equally abundant in both groups (25% in p

  14. Production of avian influenza virus vaccine using primary cell cultures generated from host organs.

    PubMed

    Babar, Mustafeez Mujtaba; Riaz, Muhammad Suleman; Zaidi, Najam-us-Sahar Sadaf; Afzal, Farhan; Farooq, Muhammad Sabir

    2013-06-01

    The global availability of a therapeutically effective influenza virus vaccine during a pandemic remains a major challenge for the biopharmaceutical industry. Long production time, coupled with decreased supply of embryonated chicken eggs (ECE), significantly affects the conventional vaccine production. Transformed cell lines have attained regulatory approvals for vaccine production. Based on the fact that the avian influenza virus would infect the cells derived from its natural host, the viral growth characteristics were studied on chicken embryo-derived primary cell cultures. The viral propagation was determined on avian origin primary cell cultures, transformed mammalian cell lines, and in ECE. A comparison was made between these systems by utilizing various cell culture-based assays. In-vitro substrate susceptibility and viral infection characteristics were evaluated by performing hemagglutination assay (HA), 50 % tissue culture infectious dose (TCID₅₀) and monitoring of cytopathic effects (CPE) caused by the virus. The primary cell culture developed from chicken embryos showed stable growth characteristics with no contamination. HA, TCID₅₀, and CPE exhibited that these cell systems were permissive to viral infection, yielding 2-10 times higher viral titer as compared to mammalian cell lines. Though the viral output from the ECE was equivalent to the chicken cell culture, the time period for achieving it was decreased to half. Some of the prerequisites of inactivated influenza virus vaccine production include generation of higher vial titer, independence from exogenous sources, and decrease in the production time lines. Based on the tests, it can be concluded that chicken embryo primary cell culture addresses these issues and can serve as a potential alternative for influenza virus vaccine production.

  15. Physical activity levels of normal-weight and overweight girls and boys during primary school recess.

    PubMed

    Stratton, Gareth; Ridgers, Nicola D; Fairclough, Stuart J; Richardson, David J

    2007-06-01

    This study aimed to compare moderate-to-vigorous physical activity (MVPA) and vigorous physical activity (VPA) in normal-weight and overweight boys and girls during school recess. Four hundred twenty children, age 6 to 10 years, were randomly selected from 25 schools in England. Three hundred seventy-seven children completed the study. BMI was calculated from height and weight measurements, and heart rate reserve thresholds of 50% and 75% reflected children's engagement in MVPA and VPA, respectively. There was a significant main effect for sex and a significant interaction between BMI category and sex for the percent of recess time spent in MVPA and VPA. Normal-weight girls were the least active group, compared with overweight boys and girls who were equally active. Fifty-one boys and 24 girls of normal weight achieved the 40% threshold; of these, 30 boys and 10 girls exceeded 50% of recess time in MVPA. Eighteen overweight boys and 22 overweight girls exceeded the 40% threshold, whereas 8 boys and 8 girls exceeded the 50% threshold. Overweight boys were significantly less active than their normal-weight male counterparts; this difference did not hold true for girls. Even though nearly double the number of normal-weight children achieved the 40% of MVPA during recess compared with overweight children, physical activity promotion in school playgrounds needs to be targeted not only at overweight but at other health parameters, as 40 overweight children met the 40% MVPA target proposed for recess.

  16. Nurses' perceptions of workplace culture in primary health care in Finland.

    PubMed

    Hahtela, N; Paavilainen, E; McCormack, B; Helminen, M; Slater, P; Suominen, T

    2015-12-01

    This study aimed to describe nurses' perceptions of workplace culture, especially in regard to stress levels, job satisfaction and the practice environment in primary health care. Health care is facing many challenges related to its attractiveness as a place of employment and the maintenance of a sufficient workforce supply. Previous studies report increasing rates of nurse job dissatisfaction and intentions to leave their current positions both in Finland and also globally. Improving workplace culture is thus vital in meeting the challenges related to recruitment and retention. A cross-sectional descriptive design was used to describe nurses' perceptions of workplace culture. Data were collected by questionnaire from 22 units in nine primary healthcare organizations in Finland, and analysed using descriptive and inferential statistics. Most of the respondents indicated that they were not certain whether their workplace culture was either positive or negative. Profession, age and work shift characteristics had an effect on the respondents' perceptions of workplace culture. Younger licensed practical and registered nurses assessed their workplace culture more positively, whereas older registered nurses and those working rotating rosters viewed workplace culture more negatively. The findings suggest that both unit and demographic characteristics affect workplace culture. This survey highlights that a positive workplace culture is one of the key factors in retaining and recruiting nurses, and provides an essential evidence that may be considered by other healthcare organizations. Nurse managers and healthcare leaders need to address workload management and take into account the related variables that affect a unit's workplace culture. © 2015 International Council of Nurses.

  17. Knowing in Primary Physical Education in the UK: Negotiating Movement Culture

    ERIC Educational Resources Information Center

    Ward, Gavin; Quennerstedt, Mikael

    2015-01-01

    This paper aims to understand how pupils and teachers actions-in-context constitute being-a-pupil and being-a-teacher within a primary school physical education (PE) movement culture. Dewey and Bentley's theory of transaction, which views organism-in-environment-as-a-whole, enables the researcher to explore how actions-in-ongoing activities…

  18. Resource and Production, A Primary Unit in Cultural Geography. Pupil Text and Workbook and Teacher Manual.

    ERIC Educational Resources Information Center

    Imperatore, William

    This is an instructional unit in cultural geography for the primary grades. The major objective of the unit, which is comprised of a Pupil Text/Workbook and Teacher Manual, is to develop the geographic concepts labeled resource and production. Teaching strategies used include the Pestalozzian method of asking leading questions to draw the students…

  19. Cationic Amino Acid Based Lipids as Effective Nonviral Gene Delivery Vectors for Primary Cultured Neurons

    PubMed Central

    2013-01-01

    The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C3-Glu2C14, and a lysine-based lipid, Lys-C3-Glu2C14, with two different types of counterion, chloride ion (Cl–) and trifluoroacetic acid (TFA–), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C3-Glu2C14 with TFA– induced a rate of transfection in primary cultured neurons higher than that of Lys-C3-Glu2C14 using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C3-Glu2C14 with TFA– showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C3-Glu2C14 with TFA– is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity. PMID:24087930

  20. Cultures of Engagement in Challenging Circumstances: Four Lebanese Primary Schools in Urban Beirut

    ERIC Educational Resources Information Center

    Nabhani, Mona; Busher, Hugh; Bahous, Rima

    2012-01-01

    This study of four private primary schools in Beirut, Lebanon, investigated why the children in the schools appeared to out-perform their peers in other schools. The study investigated the cultures that teachers and principals constructed in schools with children and their parents, wondering whether they would exhibit characteristics said to…

  1. Resource and Production, A Primary Unit in Cultural Geography. Pupil Text and Workbook and Teacher Manual.

    ERIC Educational Resources Information Center

    Imperatore, William

    This is an instructional unit in cultural geography for the primary grades. The major objective of the unit, which is comprised of a Pupil Text/Workbook and Teacher Manual, is to develop the geographic concepts labeled resource and production. Teaching strategies used include the Pestalozzian method of asking leading questions to draw the students…

  2. Promoting Cultural Understandings through Song across the Tasman: Pre-Service Primary Teacher Education

    ERIC Educational Resources Information Center

    Joseph, Dawn; Trinick, Robyn

    2016-01-01

    As tertiary music educators across the Tasman we argue that music, particularly song, is an effective medium for teaching and learning about non-western music when preparing generalist primary Pre-Service Teachers (PSTs). Using "voice" as a portable and accessible vehicle to transmit cultural understandings, we draw on the Zimbabwean…

  3. Culturally Relevant Literature: What Matters Most to Primary-Age Urban Learners

    ERIC Educational Resources Information Center

    Cartledge, Gwendolyn; Keesey, Susan; Bennett, Jessica G.; Ramnath, Rajiv; Council, Morris R., III.

    2016-01-01

    The ratings and rationales primary-age urban learners gave culturally relevant reading passages was the focus of this descriptive study. First- and second-grade students each read 30 researcher-developed passages reflecting the students' immediate and historical backgrounds. The students rated the passages and gave a reason for their ratings. A…

  4. Development of Quality Assurance System in Culture and Nation Character Education in Primary Education in Indonesia

    ERIC Educational Resources Information Center

    Susilana, Rudi; Asra

    2013-01-01

    The purpose of national education is to develop skills and build dignified national character and civilization in educating nation life (Act No. 20, 2003). The paper describes a system of quality assurance in culture and character education in primary education. This study employs the six sigma model which consists of the formula DMAIC (Define,…

  5. Promoting Cultural Understandings through Song across the Tasman: Pre-Service Primary Teacher Education

    ERIC Educational Resources Information Center

    Joseph, Dawn; Trinick, Robyn

    2016-01-01

    As tertiary music educators across the Tasman we argue that music, particularly song, is an effective medium for teaching and learning about non-western music when preparing generalist primary Pre-Service Teachers (PSTs). Using "voice" as a portable and accessible vehicle to transmit cultural understandings, we draw on the Zimbabwean…

  6. Knowing in Primary Physical Education in the UK: Negotiating Movement Culture

    ERIC Educational Resources Information Center

    Ward, Gavin; Quennerstedt, Mikael

    2015-01-01

    This paper aims to understand how pupils and teachers actions-in-context constitute being-a-pupil and being-a-teacher within a primary school physical education (PE) movement culture. Dewey and Bentley's theory of transaction, which views organism-in-environment-as-a-whole, enables the researcher to explore how actions-in-ongoing activities…

  7. Culturally Relevant Literature: What Matters Most to Primary-Age Urban Learners

    ERIC Educational Resources Information Center

    Cartledge, Gwendolyn; Keesey, Susan; Bennett, Jessica G.; Ramnath, Rajiv; Council, Morris R., III.

    2016-01-01

    The ratings and rationales primary-age urban learners gave culturally relevant reading passages was the focus of this descriptive study. First- and second-grade students each read 30 researcher-developed passages reflecting the students' immediate and historical backgrounds. The students rated the passages and gave a reason for their ratings. A…

  8. Primary-Grade Students' Knowledge and Thinking about Shelter as a Cultural Universal.

    ERIC Educational Resources Information Center

    Brophy, Jere; Alleman, Janet

    The traditional K-3 social studies curriculum has focused on food, clothing, shelter, communication, transportation, and other cultural universals. A study was designed to provide information with respect to the topic of shelter, and in the process, to assess claims that primary grade students do not need instruction in the topic because they…

  9. The role of atherosclerosis imaging in redefining normal and abnormal cholesterol values, and risk reduction in primary prevention statin trials.

    PubMed

    Hecht, Harvey S

    2011-10-01

    The emergence of atherosclerosis imaging, using coronary calcium scanning (CAC) and carotid intima media thickness (CIMT) and plaque as stronger predictors of cardiovascular events than risk factors of atherosclerosis, has created a paradigm shift in the primary prevention of cardiovascular disease. Rather than population-derived indices to define normal or abnormal low-density lipoprotein (or other lipid values) in the untreated individual patient, it is more appropriate to define "normal" as "cholesterol values at which level there is no subclinical atherosclerosis" and "abnormal" as "cholesterol values at which level there is subclinical atherosclerosis," with the severity of "abnormal" depending on the degree of subclinical atherosclerosis. Similarly, the low-density lipoprotein treatment goal is the level at which atherosclerosis progression is halted. Extension of the subclinical atherosclerosis risk-based paradigms to primary prevention trials dramatically changes the manner in which trials should be conducted in the future, as well as the results of trials already performed. For example, asymptomatic patients with a CAC score of 0 have an extraordinarily low event rate but have been included in primary prevention statin trials even though their risk without treatment is very small. Reanalysis of the statin primary prevention trials after excluding the percentage (40%) of patients who would be expected to have a CAC score of 0 yields an absolute risk increase of 60% in both the placebo group (from 5.4% to 8.5%), and the statin group (from 3.0% to 4.8%). Absolute risk reduction increased by 58% (from 2.4% to 3.8%). Relative risk reduction of 44% was unchanged. In conclusion, 1) the presence or absence of atherosclerosis as measured by CAC redefines normal or abnormal lipid levels in an individual patient; 2) statin absolute risk reduction is significantly greater than previously appreciated; and 3) patients with a CAC score of 0 should be excluded from

  10. Hyperproliferation of normally quiescent keratinocytes in non-lesional psoriatic skin due to high calcium concentration (an organotypic culture model).

    PubMed

    Szabó, Anna Kenderessy; Bos, J D; Das, P K

    2002-01-01

    Calcium plays an important role in the regulation of different functions of keratinocytes. In the present work we studied the effect of different extracellular calcium concentrations (0.01 mM-2.0 mM) on the proliferation and differentiation of human keratinocytes in normal human and non-lesional psoriatic skin. Using explant culture model, the proliferative and differentiated subsets of keratinocytes were detected by specific antibodies related to cell proliferation [beta-1 integrin (CD29), proliferating cell antigen (Ki67), proliferating cell nuclear antigen (PCNA)] and differentiation [differentiated cell cytokeratins (K1/K10) and differentiating cell antigen (lectin Ulex europaius agglutinin, UEA-1)]. After 4 days of culturing at high Ca2+ (2.0 mM) we observed marked hyperproliferation among the normally quiescent keratinocytes of non-lesional psoriatic skin. In normal uncultured and cultured skin and in uncultured and two-day-cultured non-lesional psoriatic skin both at normal (1.2 mM) and at high (2.0 mM) Ca2+ concentration only one layer of basal CD29+/Ki67+/K1/K10-/UEA-1- cell was observed. In sections from non-lesional psoriatic skin cultured for 4 days in the presence of high Ca2+ (2.0 mM) this cell population has expanded from at least three layers above the basement membrane. This expanded cell population of the 4-day high Ca2+ cultured non-lesional skin showed clear PCNA positive staining on frozen sections with the strongest positivity among the most basal localized cells. These data suggest that (i) extracellular Ca2+ concentration can influence the proliferation of basal ("stem") keratinocytes, (ii) the proliferative response to high Ca2+ concentration of psoriatic non-lesional basal keratinocytes differs from that of normal basal keratinocytes, (iv) changes in the extracellular Ca2+ milieu might play a role in the induction of the hyperproliferative psoriatic lesion.

  11. The cutaneous vasoconstrictor response to venous stasis is normal in subjects with primary Raynaud's disease.

    PubMed

    Edwards, C M; Marshall, J M; Pugh, M

    1999-10-01

    In control subjects and in subjects with primary Raynaud's disease, sudden sound or a mild cool stimulus evokes the pattern of alerting response that includes cutaneous vasoconstriction but vasodilatation in forearm muscle. In control subjects, response habituates on repetition of these stimuli both within experimental sessions and over successive days. However, in subjects with primary Raynaud's disease, the cutaneous vasoconstriction and the muscle vasodilatation persist. We have now tested whether a similar disparity exists for the cutaneous vasoconstriction evoked by venous stasis, a response considered to be a veno-arteriolar reflex mediated by sympathetic fibers, but not requiring transmission through the spinal cord. In 10 subjects with primary Raynaud's disease and in 10 matched controls, a sphygmomanometer cuff on the left arm was inflated to 40 mm Hg for 2 minutes, five times on each of three experimental sessions on days 1, 3, and 5. Cutaneous red cell flux (RCF) was recorded from the pulp and dorsum of the left index finger by using a laser Doppler meter; digital vascular conductance (DCVC) was computed as RCF divided by arterial pressure. The first venous stasis, in session 1, evoked a decrease in pulp and dorsum DCVC in the control and primary Raynaud's subjects. There were no differences between the groups in the magnitudes or durations of these responses. Within session 1, the magnitude of the decrease in DCVC diminished on repetition of venous stasis in the dorsum in controls and in the pulp in primary Raynaud's subjects. We propose these effects reflected the similar reductions in baseline DCVC over time; there was no change in the duration of the responses. Repetition of venous stasis had similar effects in both groups of subjects within sessions 2 and 3. Further, judging from the mean of the responses evoked in each Session the decreases evoked in pulp and dorsum DCVC by venous stasis were fully consistent in magnitude and duration over the

  12. Rat Sertoli cells acquire a beta-adrenergic response during primary culture.

    PubMed Central

    Kierszenbaum, A L; Spruill, W A; White, M G; Tres, L L; Perkins, J P

    1985-01-01

    Two-dimensional polyacrylamide gel electrophoresis and the radioligand (-)-[125I]iodopindolol (125I-Pin) have been used to study isoproterenol-dependent protein phosphorylation and beta-adrenergic receptor availability, respectively, in cultured Sertoli cells and freshly isolated seminiferous tubular segments of sexually immature and mature rats. Sertoli cells prepared from sexually immature rats show progressive 125I-Pin binding in primary cultures that correlates with isoproterenol-induced cell shape changes, redistribution of immunoreactive vimentin, and phosphorylation of this intermediate filament protein. The development of 125I-Pin binding to Sertoli cell lysates is blocked by cycloheximide. Seminiferous tubules do not show significant isoproterenol-dependent vimentin phosphorylation nor 125I-Pin binding. However, vimentin phosphorylation can be induced by follicle-stimulating hormone or a cyclic nucleotide analog. This study stresses the need for correlating pharmacological-induced responses observed in Sertoli cell primary cultures with those in the intact seminiferous tubule. Images PMID:2984678

  13. Molecular pathway activation features linked with transition from normal skin to primary and metastatic melanomas in human.

    PubMed

    Shepelin, Denis; Korzinkin, Mikhail; Vanyushina, Anna; Aliper, Alexander; Borisov, Nicolas; Vasilov, Raif; Zhukov, Nikolay; Sokov, Dmitry; Prassolov, Vladimir; Gaifullin, Nurshat; Zhavoronkov, Alex; Bhullar, Bhupinder; Buzdin, Anton

    2016-01-05

    Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear. For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways. We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma. Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases. We created a model describing formation and progression of melanoma at the level of molecular pathway activation. We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma). Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways. This study helps to decode molecular mechanisms underlying the development of melanoma.

  14. Molecular pathway activation features linked with transition from normal skin to primary and metastatic melanomas in human

    PubMed Central

    Shepelin, Denis; Korzinkin, Mikhail; Vanyushina, Anna; Aliper, Alexander; Borisov, Nicolas; Vasilov, Raif; Zhukov, Nikolay; Sokov, Dmitry; Prassolov, Vladimir; Gaifullin, Nurshat; Zhavoronkov, Alex; Bhullar, Bhupinder; Buzdin, Anton

    2016-01-01

    Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear. For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways. We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma. Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases. We created a model describing formation and progression of melanoma at the level of molecular pathway activation. We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma). Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways. This study helps to decode molecular mechanisms underlying the development of melanoma. PMID:26624979

  15. Detection of vital germ cell tumor cells in short-term cell cultures of primary tumors and of retroperitoneal metastasis--clinical implications.

    PubMed

    Otto, T; Virchow, S; Fuhrmann, C; Steinberg, F; Streffer, C; Goepel, M; Rübben, H

    1997-01-01

    By establishing short-term cell cultures derived from retroperitoneal metastasis after neoadjuvant chemotherapy, our aim was to improve the diagnosis and prognosis in patients with advanced testicular germ cell tumors. The histological evaluation of surgically removed metastatic tissue by retroperitoneal lymphadenectomy (RLA) is extremely complicated after previous chemotherapy, but knowledge of persistence of vital tumor cells in residual lesions is of great prognostic value and therapeutic consequence in patients with testicular germ cell tumors. We therefore investigated whether vital tumor tissue could be detected in short-term cell cultures derived from such metastatic lesions by measuring the concentration of the tumor markers beta human chorionic gonadotropin (beta HCG) and alpha-1 fetoprotein (AFP) in cell culture supernatants. We initially demonstrated the specificity of the determination in cell cultures of human transitional-cell carcinoma cell lines, human foreskin fibroblasts and normal testicular tissue. In a group of 20 patients with untreated primary testicular germ cell tumors, detection of beta HCG and AFP was increased about threefold in cell culture supernatants in comparison to the serum concentration. Finally, we prepared primary cell cultures from surgically removed retroperitoneal metastasis of 12 patients with testicular germ cell tumors after chemotherapy. The serum concentrations of beta HCG and AFP of all patients were at normal values when RLA was performed. However, pathologically increased concentrations of beta HCG (3/3) and AFP (2/3) in cell culture supernatants were found in 3 of 12 cell cultures. Interestingly, these three patients with a pathological increase in beta HCG and AFP as determined in the supernatant of the short-term cell cultures had tumor progression within a mean follow-up of 3 +/- 1 months (P < 0.01), whereas 9 of 12 patients who had no pathological increase in beta HCG and AFP as determined in the supernatant of

  16. Managing change in the culture of general practice: qualitative case studies in primary care trusts.

    PubMed

    Marshall, Martin N; Mannion, Russell; Nelson, Elizabeth; Davies, Huw T O

    2003-09-13

    To explore the potential tension between the need for managers to produce measurable change and the skills required to produce cultural change, and to investigate how managers of primary care trusts are attempting to deal with this tension. Qualitative case studies using data derived from semistructured interviews and a review of published documents. An established cultural framework was to used to help interpret the findings. Six primary care trusts in England purposefully sampled to represent a range of cultural, structural, geographical, and demographic characteristics. 42 interviews with 39 different senior and middle primary care trust managers conducted over an 18 month period. We found two distinct and polarised styles of management. One group of managers adopts a directive style and challenges the prevailing norms and values of clinicians, an approach characteristically seen in organisations with hierarchical cultures. This group is made up mostly of senior managers who are driven principally by the imperative to deliver a political agenda. Managers in the second group are more inclined to work with the prevailing cultures found in general practice, attempting to facilitate change from within rather than forcing change from outside. This management style is characteristically seen in organisations with a clan-type culture. The approach was manifest mostly by middle managers, who seem to act as buffers between the demands of senior managers and their own perception of the ability and willingness of health professionals to cope with change. The different management approaches can lead to tension and dysfunction between tiers of management. The development of primary care depends on high quality managers who are able to draw on a range of different management skills and styles. Managers are most likely to be effective if they appreciate the merits and drawbacks of their different styles and are willing to work in partnership.

  17. Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor.

    PubMed

    Vergara-Castañeda, E; Grattan, D R; Pasantes-Morales, H; Pérez-Domínguez, M; Cabrera-Reyes, E A; Morales, T; Cerbón, M

    2016-04-01

    Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model using primary cell cultures of hippocampal neurons, and to determine whether this effect is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu)--induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures.

  18. Normalizing the Fraughtness: How Emotion, Race, and School Context Complicate Cultural Competence

    ERIC Educational Resources Information Center

    Buehler, Jennifer; Ruggles Gere, Anne; Dallavis, Christian; Shaw Haviland, Victoria

    2009-01-01

    Preservice teachers seeking to develop cultural competence can face a struggle fraught with multiple challenges, even when they are committed to culturally relevant pedagogy. This article closely analyzes one White beginning teacher's negotiations with cultural competence during a lesson in her student teaching semester, then traces how she made…

  19. Normalizing the Fraughtness: How Emotion, Race, and School Context Complicate Cultural Competence

    ERIC Educational Resources Information Center

    Buehler, Jennifer; Ruggles Gere, Anne; Dallavis, Christian; Shaw Haviland, Victoria

    2009-01-01

    Preservice teachers seeking to develop cultural competence can face a struggle fraught with multiple challenges, even when they are committed to culturally relevant pedagogy. This article closely analyzes one White beginning teacher's negotiations with cultural competence during a lesson in her student teaching semester, then traces how she made…

  20. Localization of sarcomeric proteins during myofibril assembly in cultured mouse primary skeletal myotubes.

    PubMed

    White, Jennifer; Barro, Marietta V; Makarenkova, Helen P; Sanger, Joseph W; Sanger, Jean M

    2014-09-01

    It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F-actin, nonmuscle myosin II, muscle myosin II, and α-actinin were organized in the three stages of myofibril assembly. The results also test previous reports that nonmuscle myosins II A and B are components of the Z-bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z-bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP-tagged proteins to determine where and when these YFP-sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells. © 2014 Wiley Periodicals, Inc.

  1. Successful establishment of primary small airway cell cultures in human lung transplantation

    PubMed Central

    2009-01-01

    Background The study of small airway diseases such as post-transplant bronchiolitis obliterans syndrome (BOS) is hampered by the difficulty in assessing peripheral airway function either physiologically or directly. Our aims were to develop robust methods for sampling small airway epithelial cells (SAEC) and to establish submerged SAEC cultures for downstream experimentation. Methods SAEC were obtained at 62 post-transplant bronchoscopies in 26 patients using radiologically guided bronchial brushings. Submerged cell cultures were established and SAEC lineage was confirmed using expression of clara cell secretory protein (CCSP). Results The cell yield for SAEC (0.956 ± 0.063 × 106) was lower than for large airway cells (1.306 ± 0.077 × 106) but did not significantly impact on the culture establishment rate (79.0 ± 5.2% vs. 83.8 ± 4.7% p = 0.49). The presence of BOS significantly compromised culture success (independent of cell yield) for SAEC (odds ratio (95%CI) 0.067 (0.01-0.40)) but not LAEC (0.3 (0.05-1.9)). Established cultures were successfully passaged and expanded. Conclusion Primary SAEC can be successfully obtained from human lung transplant recipients and maintained in culture for downstream experimentation. This technique will facilitate the development of primary in vitro models for BOS and other diseases with a small airway component such as asthma, cystic fibrosis and COPD. PMID:19857270

  2. Circadian Rhythms of PER2::LUC in Individual Primary Mouse Hepatocytes and Cultures

    PubMed Central

    Molyneux, Penny C.; Yu, Jimmy K.; Li, Alexander S.; Leise, Tanya L.; Harrington, Mary E.

    2014-01-01

    Background Hepatocytes, the parenchymal cells of the liver, express core clock genes, such as Period2 and Cryptochrome2, which are involved in the transcriptional/translational feedback loop of the circadian clock. Whether or not the liver is capable of sustaining rhythms independent of a central pacemaker is controversial. Whether and how circadian information may be shared among cells in the liver in order to sustain oscillations is currently unknown. Results In this study we isolated primary hepatocytes from transgenic Per2Luc mice and used bioluminescence as a read-out of the state of the circadian clock. Hepatocytes cultured in a collagen gel sandwich configuration exhibited persistent circadian rhythms for several weeks. The amplitude of the rhythms damped, but medium changes consistently reset the phase and amplitude of the cultures. Cry2−/− Per2Luc cells oscillated robustly and expressed a longer period. Co-culturing with wildtype cells did not significantly shorten the period, indicating that coupling among hepatocytes is insufficient to synchronize cells with significantly differing periods. However, spatial patterns revealed by cellular imaging of wildtype cultures provided evidence of weak local coupling among the hepatocytes. Conclusions Our results with primary hepatocyte cultures demonstrate that cultured hepatocytes are weakly coupled. While this coupling is not sufficient to sustain global synchrony, it does increase local synchrony, which may stabilize the circadian rhythms of peripheral oscillators, such as the liver, against noise in the entraining signals. PMID:24498336

  3. Primary cell cultures from sea urchin ovaries: a new experimental tool.

    PubMed

    Mercurio, Silvia; Di Benedetto, Cristiano; Sugni, Michela; Candia Carnevali, M Daniela

    2014-02-01

    In the present work, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed in order to provide a simple and versatile experimental tool for researches in echinoderm reproductive biology. Ovary cell phenotypes were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in minimum essential medium Eagle and medium 199. Different substrates were tested, but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation, different serum-supplements were tested. Fetal calf serum and an originally developed pluteus extract were detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin egg extract appeared larger and healthier, displaying an increased longevity that allowed maintaining them for up to 1 month. Overall, our study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries offering a good potential to study echinoid oogenesis in a controlled system and to investigate different aspects of echinoderm endocrinology and reproductive biology.

  4. Trichostatin A, a critical factor in maintaining the functional differentiation of primary cultured rat hepatocytes

    SciTech Connect

    Henkens, Tom . E-mail: Tom.Henkens@vub.ac.be; Papeleu, Peggy; Elaut, Greetje; Vinken, Mathieu; Rogiers, Vera; Vanhaecke, Tamara

    2007-01-01

    Histone deacetylase inhibitors (HDI) have been shown to increase differentiation-related gene expression in several tumor-derived cell lines by hyperacetylating core histones. Effects of HDI on primary cultured cells, however, have hardly been investigated. In the present study, the ability of trichostatin A (TSA), a prototype hydroxamate HDI, to counteract the loss of liver-specific functions in primary rat hepatocyte cultures has been investigated. Upon exposure to TSA, it was found that the cell viability of the cultured hepatocytes and their albumin secretion as a function of culture time were increased. TSA-treated hepatocytes also better maintained cytochrome P450 (CYP)-mediated phase I biotransformation capacity, whereas the activity of phase II glutathione S-transferases (GST) was not affected. Western blot and qRT-PCR analysis of CYP1A1, CYP2B1 and CYP3A11 protein and mRNA levels, respectively, further revealed that TSA acts at the transcriptional level. In addition, protein expression levels of the liver-enriched transcription factors (LETFs) hepatic nuclear factor 4 alpha (HNF4{alpha}) and CCAAT/enhancer binding protein alpha (C/EBP{alpha}) were accordingly increased by TSA throughout culture time. In conclusion, these findings indicate that TSA plays a major role in the preservation of the differentiated hepatic phenotype in culture. It is suggested that the effects of TSA on CYP gene expression are mediated via controlling the expression of LETFs.

  5. Optic Disc Perfusion in Primary Open Angle and Normal Tension Glaucoma Eyes Using Optical Coherence Tomography-Based Microangiography

    PubMed Central

    Wen, Joanne C.; Zhang, Qinqin; Xin, Chen; Gupta, Divakar; Mudumbai, Raghu C.; Johnstone, Murray A.; Wang, Ruikang K.; Chen, Philip P.

    2016-01-01

    Purpose To investigate optic disc perfusion differences in normal, primary open-angle glaucoma (POAG), and normal tension glaucoma (NTG) eyes using optical microangiography (OMAG) based optical coherence tomography (OCT) angiography technique. Design Cross-sectional, observational study. Subjects Twenty-eight normal, 30 POAG, and 31 NTG subjects. Methods One eye from each subject was scanned with a 68 kHz Cirrus HD-OCT 5,000-based OMAG prototype system centered at the optic nerve head (ONH) (Carl Zeiss Meditec Inc, Dublin, CA). Microvascular images were generated from the OMAG dataset by detecting the differences in OCT signal between consecutive B-scans. The pre-laminar layer (preLC) was isolated by a semi-automatic segmentation program. Main Outcome Measures Optic disc perfusion, quantified as flux, vessel area density, and normalized flux (flux normalized by the vessel area) within the ONH. Results Glaucomatous eyes had significantly lower optic disc perfusion in preLC in all three perfusion metrics (p<0.0001) compared to normal eyes. The visual field (VF) mean deviation (MD) and pattern standard deviation (PSD) were similar between the POAG and NTG groups, and no differences in optic disc perfusion were observed between POAG and NTG. Univariate analysis revealed significant correlation between optic disc perfusion and VF MD, VF PSD, and rim area in both POAG and NTG groups (p≤0.0288). However, normalized optic disc perfusion was correlated with some structural measures (retinal nerve fiber layer thickness and ONH cup/disc ratio) only in POAG eyes. Conclusions Optic disc perfusion detected with OMAG was significantly reduced in POAG and NTG groups compared to normal controls, but no difference was seen between POAG and NTG groups with similar levels of VF damage. Disc perfusion was significantly correlated with VF MD, VF PSD, and rim area in glaucomatous eyes. Vascular changes at the optic disc as measured using OMAG may provide useful information for

  6. Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions.

    PubMed

    Lee, Sook-Jeong; Park, Mi-Ha; Kim, Hyun-Jae; Koh, Jae-Young

    2010-08-01

    Cellular zinc plays a key role in lysosomal change and cell death in neurons and astrocytes under oxidative stress. Here, using astrocytes lacking metallothionein-3 (MT3), a potential source of labile zinc in the brain, we studied the role of MT3 in oxidative stress responses. H(2)O(2) induced a large increase in labile zinc in wild-type (WT) astrocytes, but stimulated only a modest rise in MT3-null astrocytes. In addition, H(2)O(2)-induced lysosomal membrane permeabilization (LMP) and cell death were comparably attenuated in MT3-null astrocytes. Expression and glycosylation of Lamp1 (lysosome-associated membrane protein 1) and Lamp2 were increased in MT3-null astrocytes, and the activities of several lysosomal enzymes were significantly reduced, indicating an effect of MT3 on lysosomal components. Consistent with lysosomal dysfunction in MT3-null cells, the level of LC3-II (microtubule-associated protein 1 light chain 3), a marker of early autophagy, was increased by oxidative stress in WT astrocytes, but not in MT3-null cells. Similar changes in Lamp1, LC3, and cathepsin-D were induced by the lysosomal inhibitors bafilomycin A1, chloroquine, and monensin, indicating that lysosomal dysfunction may lie upstream of changes observed in MT3-null astrocytes. Consistent with this idea, lysosomal accumulation of cholesterol and lipofuscin were augmented in MT3-null astrocytes. Similar to the results seen in MT3-null cells, MT3 knockdown by siRNA inhibited oxidative stress-induced increases in zinc and LMP. These results indicate that MT3 may play a key role in normal lysosomal function in cultured astrocytes.

  7. Fibrin promotes development and function of macaque primary follicles during encapsulated three-dimensional culture.

    PubMed

    Xu, J; Lawson, M S; Yeoman, R R; Molskness, T A; Ting, A Y; Stouffer, R L; Zelinski, M B

    2013-08-01

    Does fibrin introduced into the extracellular matrix affect the growth and maturation of individual primate follicles during encapsulated three-dimensional (3D) culture? While not altering follicle survival, fibrin-alginate (FIBRIN) improves macaque primary, but not secondary, follicle development during encapsulated 3D culture in terms of growth, steroidogenesis, anti-Müllerian hormone (AMH)/vascular endothelial growth factor (VEGF) production and oocyte maturation. Efforts to grow non-human primate ovarian follicles from the secondary to the antral stage during encapsulated 3D culture have been successful. However, the growth and maturation of primary follicles in vitro has not been reported in primates, especially in chemically defined conditions. In vitro follicle maturation was investigated using the rhesus macaque (Macaca mulatta). Ovaries (n = 7 pairs) were obtained during the early follicular phase of the menstrual cycle (cycle day 1-4). Primary (80-120 µm diameter) and secondary (125-225 µm diameter) follicles were isolated mechanically, randomly assigned to experimental groups, encapsulated into alginate (0.25% w/v) or FIBRIN (25 mg/ml fibrinogen-0.25% alginate) and cultured for 13 and 5 weeks, respectively. Individual follicles were cultured in alpha minimum essential medium supplemented with FSH. Follicle survival and growth were assessed by microscopy. Follicles that reached the antral stage were treated with recombinant hCG. Metaphase II (MII) oocytes were inseminated via ICSI. Follicle morphology was evaluated by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed for cytochrome P450 family 17 subfamily A polypeptide 1 (CYP17A1) and 19 subfamily A polypeptide 1 (CYP19A1). Culture medium was analyzed for estradiol (E2) and progesterone by chemiluminescence, androstenedione (A4) by radioimmunoassay, as well as anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay. A

  8. Early oxidative damage in primary cultured trout hepatocytes: a time course study.

    PubMed

    Ferraris, Michela; Radice, Sonia; Catalani, Paolo; Francolini, Maura; Marabini, Laura; Chiesara, Enzo

    2002-09-24

    The aim of this study was to evaluate the influence of the two-step hepatocyte isolation procedure on primary cultured trout (Oncorhynchus mykiss) hepatocytes over time. We characterised the possible changes of a variety of some cellular parameters within the first 24-48 h after seeding. We followed the time dependent changes of these parameters during subsequent culture times in order to see if the cells maintained a differentiated status. Scanning electron microscopy revealed bleb formation and 20% cell damage in freshly isolated hepatocytes. During subsequent culture times the bleb dimension appear to be reduced. Heat shock proteins 70 and 50 (HSP70, HSP50) were induced by hepatocyte isolation. During the first 4 h of culture, the hepatocytes showed a variation in mitochondrial activity, an increase in free radical species (ROS), and a decrease in both glutathione (GSH) content and catalase (CAT) activity; the generation of free radicals led to an increase in the formation of 8-hydroxydeoxyguanosine (8-OHdG) in the DNA. The cells showed detectable ethoxyresorufin-O-deethylase activity after 4 h of culture, which had rapidly increased by the 24th hour. After 24 h, mitochondrial and CAT activity, free radical production, and the content of GSH and 8-OHdG returned to their original levels. P450 activity was retained for at least 48 h after seeding. Our data show that trout hepatocytes suffer significant cell injury as a result of the isolation procedure, but primary cultured cells metabolically recover from this stress after a few hours: they are capable of repairing their damaged surfaces, recovering their antioxidant defences and retaining their ability to repair DNA. Our results also confirm that trout hepatocytes in a primary culture maintain their in vivo-like metabolic activities for 3-8 days.

  9. Picture superiority in free recall: the effects of normal aging and primary degenerative dementia.

    PubMed

    Rissenberg, M; Glanzer, M

    1986-01-01

    A key factor in the decline of memory with age may be a breakdown of communication in the information network involved in memory and cognitive processing. A special case of this communication is assumed to underlie the picture superiority effect in recall. From this hypothesis it follows that the picture superiority effect should lessen with age. In Experiment 1, three groups of adults (young, old normal, and old memory-impaired) were tested in free recall of pictures and word lists. As predicted, the picture superiority effect declined with age. Experiment 2 replicated these findings and showed, moreover, that the picture superiority effect can be reestablished in normal old adults by instructing them to verbalize overtly during item presentation.

  10. Crouzon syndrome with primary optic nerve atrophy and normal brain functions: A case report.

    PubMed

    Pal, Uma Shankar; Gupta, Chandan; Chellappa, Arul A L

    2012-01-01

    This report and review of literature aimed to assess an unusual case of Crouzon syndrome characterized by distinctive disfigurement of craniofacial skeletal and soft tissue structures with primary optic nerve atropy. We present a case of a 12-year-old girl with Crouzon syndrome displaying classic facial abnormalities with reduced vision and hearing loss. Crouzon syndrome should be managed as early as possible as it results in airway obstruction, decreased vision, mental retardation and poor cosmetic appearance.

  11. Crouzon syndrome with primary optic nerve atrophy and normal brain functions: A case report

    PubMed Central

    Pal, Uma Shankar; Gupta, Chandan; Chellappa, Arul A.L.

    2012-01-01

    Background This report and review of literature aimed to assess an unusual case of Crouzon syndrome characterized by distinctive disfigurement of craniofacial skeletal and soft tissue structures with primary optic nerve atropy. Methods We present a case of a 12-year-old girl with Crouzon syndrome displaying classic facial abnormalities with reduced vision and hearing loss. Conclusion Crouzon syndrome should be managed as early as possible as it results in airway obstruction, decreased vision, mental retardation and poor cosmetic appearance. PMID:25737846

  12. Mitochondrial respiratory chain dysfunction modulates metalloproteases -1, -3 and -13 in human normal chondrocytes in culture.

    PubMed

    Cillero-Pastor, Berta; Rego-Pérez, Ignacio; Oreiro, Natividad; Fernandez-Lopez, Carlos; Blanco, Francisco J

    2013-08-09

    Mitochondrion has an important role in the osteoarthritis (OA) pathology. We have previously demonstrated that the alteration of the mitochondrial respiratory chain (MRC) contributes to the inflammatory response of the chondrocyte. However its implication in the process of cartilage destruction is not well understood yet. In this study we have investigated the relationship between the MRC dysfunction and the regulation of metalloproteases (MMPs) in human normal chondrocytes in culture. Human normal chondrocytes were isolated from human knees obtained form autopsies of donors without previous history of rheumatic disease. Rotenone, 3-Nitropropionic acid (NPA), Antimycin A (AA), Sodium azide and Oligomycin were used to inhibit the activity of the mitochondrial complexes I, II, III, IV and V respectively. The mRNA expression of MMPs -1, -3 and -13 was studied by real time PCR. The intracellular presence of MMP proteins was evaluated by western blot. The liberation of these proteins to the extracellular media was evaluated by ELISA. The presence of proteoglycans in tissue was performed with tolouidin blue and safranin/fast green. Immunohistochemistry was used for evaluating MMPs on tissue. Firstly, cells were treated with the inhibitors of the MRC for 24 hours and mRNA expression was evaluated. An up regulation of MMP-1 and -3 mRNA levels was observed after the treatment with Oligomycin 5 and 100 μg/ml (inhibitor of the complex V) for 24 hours. MMP-13 mRNA expression was reduced after the incubation with AA 20 and 60 μg/ml (inhibitor of complex III) and Oligomycin. Results were validated at protein level observing an increase in the intracellular levels of MMP-1 and -3 after Oligomycin 25 μg/ml stimulation [(15.20±8.46 and 4.59±1.83 vs. basal=1, respectively (n=4; *P<0.05)]. However, AA and Oligomycin reduced the intracellular levels of the MMP-13 protein (0.70±0.16 and 0.3±0.24, respectively vs. basal=1). In order to know whether the MRC dysfunction had an

  13. Mitochondrial respiratory chain dysfunction modulates metalloproteases -1, -3 and -13 in human normal chondrocytes in culture

    PubMed Central

    2013-01-01

    Background Mitochondrion has an important role in the osteoarthritis (OA) pathology. We have previously demonstrated that the alteration of the mitochondrial respiratory chain (MRC) contributes to the inflammatory response of the chondrocyte. However its implication in the process of cartilage destruction is not well understood yet. In this study we have investigated the relationship between the MRC dysfunction and the regulation of metalloproteases (MMPs) in human normal chondrocytes in culture. Methods Human normal chondrocytes were isolated from human knees obtained form autopsies of donors without previous history of rheumatic disease. Rotenone, 3-Nitropropionic acid (NPA), Antimycin A (AA), Sodium azide and Oligomycin were used to inhibit the activity of the mitochondrial complexes I, II, III, IV and V respectively. The mRNA expression of MMPs -1, -3 and -13 was studied by real time PCR. The intracellular presence of MMP proteins was evaluated by western blot. The liberation of these proteins to the extracellular media was evaluated by ELISA. The presence of proteoglycans in tissue was performed with tolouidin blue and safranin/fast green. Immunohistochemistry was used for evaluating MMPs on tissue. Results Firstly, cells were treated with the inhibitors of the MRC for 24 hours and mRNA expression was evaluated. An up regulation of MMP-1 and -3 mRNA levels was observed after the treatment with Oligomycin 5 and 100 μg/ml (inhibitor of the complex V) for 24 hours. MMP-13 mRNA expression was reduced after the incubation with AA 20 and 60 μg/ml (inhibitor of complex III) and Oligomycin. Results were validated at protein level observing an increase in the intracellular levels of MMP-1 and -3 after Oligomycin 25 μg/ml stimulation [(15.20±8.46 and 4.59±1.83 vs. basal=1, respectively (n=4; *P<0.05)]. However, AA and Oligomycin reduced the intracellular levels of the MMP-13 protein (0.70±0.16 and 0.3±0.24, respectively vs. basal=1). In order to know whether the

  14. Raman spectroscopy analysis of differences in composition of spent culture media of in vitro cultured preimplantation embryos isolated from normal and fat mice dams.

    PubMed

    Fabian, Dušan; Kačmarová, Martina; Kubandová, Janka; Čikoš, Štefan; Koppel, Juraj

    2016-06-01

    The aim of the present study was to compare overall patterns of metabolic activity of in vitro cultured preimplantation embryos isolated from normal and fat mice dams by means of non-invasive profiling of spent culture media using Raman spectroscopy. To produce females with two different types of body condition (normal and fat), a previously established two-generation model was used, based on overfeeding of experimental mice during prenatal and early postnatal development. Embryos were isolated from spontaneously ovulating and naturally fertilized dams at the 2-cell stage of development and cultured to the blastocyst stage in synthetic oviductal medium KSOMaa. Embryos from fat mice (displaying significantly elevated body weight and fat) showed similar developmental capabilities in vitro as embryos isolated from normal control dams (displaying physiological body weight and fat). The results show that alterations in the composition of culture medium caused by the presence of developing mouse preimplantation embryos can be detected using Raman spectroscopy. Metabolic activity of embryos was reflected in evident changes in numerous band intensities in the 1620-1690cm(-1) (amide I) region and in the 1020-1140cm(-1) region of the Raman spectrum for KSOMaa. Moreover, multivariate analysis of spectral data proved that the composition of proteins and other organic compounds in spent samples obtained after the culture of embryos isolated from fat dams was different from that in spent samples obtained after the culture of embryos from control dams. This study demonstrates that metabolic activity of cultured preimplantation embryos might depend on the body condition of their donors.

  15. Isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains.

    PubMed

    Weinert, Maria; Selvakumar, Tharakeswari; Tierney, Travis S; Alavian, Kambiz N

    2015-02-19

    Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson's diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

  16. A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture.

    PubMed

    Lu, Zhongming; Piechowicz, Mariel; Qiu, Shenfeng

    2016-03-05

    Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (>3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for >3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

  17. Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.

    PubMed

    Esch, Mandy B; Prot, Jean-Matthieu; Wang, Ying I; Miller, Paula; Llamas-Vidales, Jose Ricardo; Naughton, Brian A; Applegate, Dawn R; Shuler, Michael L

    2015-05-21

    We have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop between reservoirs and the accompanying periodically changing fluidic flow (average flow rate of 650 μL min(-1), and a maximum shear stress of 0.64 dyne cm(-2)). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures increase their metabolic activity in response to fluidic flow periodically changes direction. Since fluidic flow that changes direction periodically drastically changes the behavior of other cells types that are shear sensitive, our findings support the theory that the increase in hepatic metabolic activity associated with fluidic flow is either activated by mechanisms other than shear sensing (for example increased opportunities for gas and metabolite exchange), or that it follows a shear sensing mechanism that does not depend on the direction of shear. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation

  18. [Nutritional analysis of dietary patterns in students of primary education with normal nutritional status].

    PubMed

    Durá-Gúrpide, Beatriz; Durá-Travé, Teodoro

    2014-06-01

    Objetivo: Realizar un análisis nutricional del modelo dietético en un grupo de alumnos de Educación Primaria (9-12 años) con estado nutricional normal. Material y Métodos: Registro de consumo de alimentos de dos días lectivos consecutivos en una muestra de 353 alumnos de Educación Primaria (188 varones y 165 mujeres) con una situación nutricional normal. Se ha calculado el consumo calórico y de macronutrientes, minerales y vitaminas comparándose con las ingestas recomendadas. Resultados: El valor medio del aporte calórico diario era de 2.066,9 kcal. Los cereales (33%), lácteos (19%) y carnes (17%) aportaban el 70% de la ingesta calórica total. Las proteínas aportaban el 20,3% de la ingesta calórica, los glúcidos el 48,8%, los lípidos el 30,9%, y las grasas saturadas el 12,6%. La ingesta de colesterol era excesiva y 2/3 de la ingesta proteica eran de origen animal. El valor medio de la ingesta de calcio, yodo y vitaminas A, D y E eran inferiores a los aportes dietéticos recomendados. Conclusiones: El modelo dietético de los alumnos de Educación Primaria con estado nutricional normal difiere del prototipo mediterráneo, con un consumo excesivo de carnes, limitado de cereales y lácteos, y deficiente en verduras y hortalizas, frutas, legumbres y pescados; dando lugar a un incremento del aporte de proteínas y grasas animales en detrimento de los hidratos de carbono complejos y un aporte deficiente de calcio, yodo y vitaminas A, D y E.

  19. Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

    PubMed Central

    Meucci, Olimpia

    2011-01-01

    This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons. Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection. A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1. At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do

  20. Basement Membrane Mimics of Biofunctionalized Nanofibers for a Bipolar-Cultured Human Primary Alveolar-Capillary Barrier Model.

    PubMed

    Nishiguchi, Akihiro; Singh, Smriti; Wessling, Matthias; Kirkpatrick, Charles J; Möller, Martin

    2017-03-13

    In vitro reconstruction of an alveolar barrier for modeling normal lung functions and pathological events serve as reproducible, high-throughput pharmaceutical platforms for drug discovery, diagnosis, and regenerative medicine. Despite much effort, the reconstruction of organ-level alveolar barrier functions has failed due to the lack of structural similarity to the natural basement membrane, functionalization with specific ligands for alveolar cell function, the use of primary cells and biodegradability. Here we report a bipolar cultured alveolar-capillary barrier model of human primary cells supported by a basement membrane mimics of fully synthetic bifunctional nanofibers. One-step electrospinning process using a bioresorbable polyester and multifunctional star-shaped polyethylene glycols (sPEG) enables the fabrication of an ultrathin nanofiber mesh with interconnected pores. The nanofiber mesh possessed mechanical stability against cyclic expansion as seen in the lung in vivo. The sPEGs as an additive provide biofunctionality to fibers through the conjugation of peptide to the nanofibers and hydrophilization to prevent unspecific protein adsorption. Biofunctionalized nanofiber meshes facilitated bipolar cultivation of endothelial and epithelial cells with fundamental alveolar functionality and showed higher permeability for molecules compared to microporous films. This nanofiber mesh for a bipolar cultured barrier have the potential to promote growth of an organ-level barrier model for modeling pathological conditions and evaluating drug efficacy, environmental pollutants, and nanotoxicology.

  1. Reactivity of alveolar epithelial cells in primary culture with type I cell monoclonal antibodies.

    PubMed

    Danto, S I; Zabski, S M; Crandall, E D

    1992-03-01

    An understanding of the process of alveolar epithelial cell growth and differentiation requires the ability to trace and analyze the phenotypic transitions that the cells undergo. This analysis demands specific phenotypic probes to type II and, especially, type I pneumocytes. To this end, monoclonal antibodies have been generated to type I alveolar epithelial cells using an approach designed to enhance production of lung-specific clones from a crude lung membrane preparation. The monoclonal antibodies were screened by a combination of enzyme-linked immunosorbent assay and immunohistochemical techniques, with the determination of type I cell specificity resting primarily on immunoelectron microscopic localization. Two of these new markers of the type I pneumocyte phenotype (II F1 and VIII B2) were used to analyze primary cultures of type II cells growing on standard tissue culture plastic and on a variety of substrata reported to affect the morphology of these cells in culture. On tissue culture plastic, the antibodies fail to react with early (days 1 to 3) type II cell cultures. The cells become progressively more reactive with time in culture to a plateau of approximately 6 times background by day 8, with a maximum rate of increase between days 3 and 5. This finding is consistent with the hypothesis that type II cells in primary culture undergo at least partial differentiation into type I cells. Type II cells grown on laminin, which reportedly delays the loss of type II cell appearance, and on fibronectin, which has been reported to facilitate cell spreading and loss of type II cell features, develop the type I cell markers during cultivation in vitro with kinetics similar to those on uncoated tissue culture plastic. Cells on type I collagen and on tissue culture-treated Nuclepore filters, which have been reported to support monolayers with type I cell-like morphology, also increase their expression of the II F1 and VIII B2 epitopes around days 3 to 5. Taken

  2. Exploring processes of organization of normal and neoplastic epithelial tissues in gradient culture.

    PubMed

    Leighton, J

    1994-09-01

    The biology of animal cells in culture is often studied in individual cells or in sheets of cells. The relevance of such studies to the intact animal is unclear, since the spatial conditions encountered by cells in animals is one of dense three-dimensional masses of cells, with limits to migration, and with gradients both of diffusion of metabolites and of morphologic maturation. These spatial requisites have gradually been met in culture. A brief account describes sponge matrix culture for three-dimensional growth and unilaminar, bilaminar, and radial histophysiologic gradient cultures. Some of the common neoplastic abnormalities of surface epithelial tissues are considered. Proposals for investigating the histokinetic mechanisms regulating some epithelial tissue processes are suggested. In the most recent development of gradient culture methods, a thin permeable transparent collagen membrane is intrinsically strengthened by producing a waffle membrane pattern for histophysiologic gradient culture.

  3. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    PubMed

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  4. Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

    PubMed Central

    Gelfand, Vladimir I.

    2013-01-01

    Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport. PMID:24300413

  5. Isolation and primary culture of gill and digestive gland cells from the common mussel Mytilus edulis.

    PubMed

    Faucet, Jérôme; Maurice, Manuelle; Gagnaire, Béatrice; Renault, Tristan; Burgeot, Thierry

    2003-01-01

    As the marine mussel Mytilus edulis is commonly used as a sentinel species, it would be useful to develop a primary culture of the target organs most often in contact with the marine environment. This study reports an improved method for dissociating the digestive gland and gills of M. edulis and considers the effect of mussel storage on cell viability and functionality before culture initiation. Viability and enzymatic activities such as those of esterase and peroxidase were monitored by flow cytometry, a sensitive, objective technique allowing large volumes of cells to be counted within a short time. A primary culture of digestive gland showed more than 75% viability after 72 h. Mussels were maintained in an aquarium containing clean, oxygenated seawater at 12 degrees C for two days before culture initiation, and dissociation was performed mechanically and chemically with Ca-Mg-free saline to obtain digestive gland cells. Application of non-specific esterase activity, using fluorescein diacetate (FDA test) coupled with flow cytometry, characterised the functionality of digestive gland and gill cells in culture.

  6. Organelle transport in cultured Drosophila cells: S2 cell line and primary neurons.

    PubMed

    Lu, Wen; Del Castillo, Urko; Gelfand, Vladimir I

    2013-11-20

    Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

  7. Hydrocortisone effect of arylsulfatase A in primary mouse brain cell cultures

    SciTech Connect

    Marcelo, A.; Pieringer, R.A.

    1986-05-01

    The primary goal of this study was to study the mechanism of action of hydrocortisone (HC) on arylsulfatase A (ASA) in primary cultures of cells that were dissociated from the brains of embryonic mice. Cells were cultured in a defined medium in the absence or in the presence of 3 ..mu..M HC. The specific activity of ASA in nontreated cells was 1.297 U/mg (U = ..mu..mol/hr) while the value for the HC-treated cells was 0.783 U/mg. The authors data shows that HC inhibits ASA activity in these cultures cells (p < 0.001). The determination of the ASA enzyme activity was assayed primarily with the artificial substrate p-nitrocatechol sulfate. However, the natural substrate (cerebroside /sup 35/S-sulfate) also as active and correlated linearly with the activity of p-nitrocatechol sulfate. Purified ASA was isolated from calf brains and used to generate an antibody (Ab) against ASA. The specificity of the Ab for the ASA protein of cell cultures was tested in Ouchterlony double immunodiffusion studies. The Ab was used in a competitive enzyme-linked immunosorbent assay to quantify the number of ASA molecules in the cell extracts from the embryonic mouse cell cultures. Preliminary data suggest that HC decreases the number of ASA molecules.

  8. Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

    PubMed Central

    Huang, Zhengmin; Harata, N. Charles

    2015-01-01

    High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences. PMID:25742545

  9. Prostaglandin E2 synthesis by human primary and metastatic bone tumors in culture.

    PubMed

    Gebhardt, M C; Lippiello, L; Bringhurst, F R; Mankin, H J

    1985-06-01

    Prostaglandin E2 (PGE2) is known to stimulate osteolysis in vitro and has been implicated in mediating bone resorption in several animal and human tumors. Little attention has yet to be directed toward local humoral control (including PGE2) of bone resorption in primary and metastatic bone tumors. For investigation of whether histologically identified areas of osteolytic or osteoblastic bone tumors differentially secrete PGE2 under in vitro conditions, culture media from explants of central and peripheral areas of tissue were sterilely collected from 13 surgical specimens of primary and metastatic bone tumors and assayed for (PGE2) radioimmunoassay. The results indicate a marked heterogeneity in the concentration of immunoreactive (I-PGE2) synthesis by tumors of different as well as similar cell type. PGE2 production was time-dependent in culture, and at 72 hours substantial increases were apparent compared to cultures of non-neoplastic fascia controls. Significantly higher levels of I-PGE2 were found in cultures derived from "bone-destructive" tumors. No difference in I-PGE2 synthesis was found between explants of peripheral versus central tissue of the same tumors. PGE2 is synthesized in culture by bone tumors characterized as destructive of bone at higher levels than "bone-forming" tumors, and this synthesis is inhibited by indomethacin.

  10. Nanofabricated Collagen-Inspired Synthetic Elastomers for Primary Rat Hepatocyte Culture

    PubMed Central

    Bettinger, Christopher J.; Kulig, Katherine M.; Vacanti, Joseph P.

    2009-01-01

    Synthetic substrates that mimic the properties of extracellular matrix proteins hold significant promise for use in systems designed for tissue engineering applications. In this report, we designed a synthetic polymeric substrate that is intended to mimic chemical, mechanical, and topological characteristics of collagen. We found that elastomeric poly(ester amide) substrates modified with replica-molded nanotopographic features enhanced initial attachment, spreading, and adhesion of primary rat hepatocytes. Further, hepatocytes cultured on nanotopographic substrates also demonstrated reduced albumin secretion and urea synthesis, which is indicative of strongly adherent hepatocytes. These results suggest that these engineered substrates can function as synthetic collagen analogs for in vitro cell culture. PMID:18847357

  11. Plaque morphology of Teschen disease viruses and certain pig enteroviruses in primary pig kidney monolayer cultures.

    PubMed

    Dardiri, A H

    1968-04-01

    Plaque patterns and diameters of four virulent strains and one tissue culture mutant of Teschen disease virus were compared with six pig enteroviruses isolated in the United States. They are described as they were produced in primary pig kidney monolayer cultures. Reproducible plaques, with similar characteristics and class-types of each of the viruses tested were obtained with the application of a 45-minute virus adsorption time. Their morphologic characteristics and the proportion in which the plaque types appeared may assist in the differentiation of these virus strains.

  12. Comparative Metabolism, Cytotoxicity, and Genotoxicity of Chemical Carcinogens in Primary Cultures of Hepatocytes

    DTIC Science & Technology

    1985-05-01

    death and regeneration. Cytotoxicity is easily assessed in primary hepatocyte cultures by light microscopic observation (Green ott al., 1981) and/or the...The recent development of a sensitive scintillation detection of repair provides a three day assay (Loury and Byard, 1983). A UV light positive control...RATS FED 0.5% BUTYLATED HYDROXYTOLUENEa Amount of ascron.•,cut./cultur-b P10o0. Al’"I boumd/t ,acrtmooletuloc Ratlo of bound producto k’ontro I WN

  13. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    SciTech Connect

    Miyoshi, Ko; Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato

    2009-10-30

    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li{sub 2}CO{sub 3} were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  14. Age- and Gender-Normalized Coronary Incidence and Mortality Risks in Primary and Secondary Prevention

    PubMed Central

    Puddu, Paolo Emilio; Iannetta, Loredana; Schiariti, Michele

    2012-01-01

    Epidemiologic differences in ischemic heart disease incidence between women and men remain largely unexplained. The reasons of women’s “protection” against coronary artery disease (CAD) are not still clear. However, there are subsets more likely to die of a first myocardial infarction. The purpose of this review is to underline different treatment strategies between genders and describe the role of classical and novel factors defined to evaluate CAD risk and mortality, aimed at assessing applicability and relevance for primary and secondary prevention. Women and men present different age-related risk patterns: it should be important to understand whether standard factors may index CAD risk, including mortality, in different ways and/or whether specific factors might be targeted gender-wise. Take home messages include: HDL-cholesterol levels, higher in pre-menopausal women than in men, are more strictly related to CAD. The same is true for high triglycerides and Lp(a). HDL-cholesterol levels are inversely related to incidence and mortality. In primary prevention the role of statins is not completely ascertained in women although in secondary prevention these agents are equally effective in both genders. Weight and glycemic control are effective to reduce cardiovascular disease (CVD) mortality in women from middle to older age. Blood pressure is strongly and directly related to CVD mortality, from middle to older age, particularly in diabetic and over weighted women. Kidney dysfunction, defined using UAE and eGFR predicts primary CVD incidence and risk in both genders. In secondary prediction, kidney dysfunction predicts sudden death in women in conjunction with left ventricular ejection fraction evaluation. Serum uric acid does not differentiate gender-related CVD incidences, although it increases with age. Age-related differences between genders have been related to loss of ovarian function traditionally and to lower iron stores more recently. QT interval

  15. Inhibition of collagen production in scleroderma fibroblast cultures by a connective tissue glycoprotein extracted from normal dermis

    SciTech Connect

    Maquart, F.X.; Bellon, G.; Cornillet-Stoupy, J.; Randoux, A.; Triller, R.; Kalis, B.; Borel, J.P.

    1985-08-01

    It was shown in a previous paper that a connective tissue glycoprotein (CTGP) extracted from normal rabbit dermis was able to inhibit total protein and collagen syntheses by normal dermis fibroblast cultures. In the present study, the effects of CTGP on scleroderma fibroblasts were investigated. (/sup 14/C)Proline incorporation into total proteins of the supernatant was not significantly different from that found in controls. By contrast, the amount of collagen, expressed as percentage of total secreted protein, was far higher in scleroderma cultures than in normal ones (14.4% +/- 6.0% vs 4.6% +/- 0.9%). Addition of CTGP to the medium induced a concentration-dependent inhibition of (/sup 14/C)proline incorporation into proteins from both control and scleroderma cells. In control cultures, no significant decrease of the percentage of collagen was observed, but over 60 micrograms/ml, both cytotoxic effects and inhibition of protein synthesis occurred. In scleroderma cultures, the inhibition was twice as effective on collagen as on noncollagen protein synthesis. The inhibition of collagen secretion was not related either to changes in collagen hydroxylation or to the intracellular catabolism of newly synthesized procollagen.

  16. Impact of Co-Culturing with Fractionated Carbon-Ion-Irradiated Cancer Cells on Bystander Normal Cells and Their Progeny.

    PubMed

    Autsavapromporn, Narongchai; Liu, Cuihua; Konishi, Teruaki

    2017-09-01

    The purpose of this study was to compare the biological effects of fractionated doses versus a single dose of high-LET carbon ions in bystander normal cells, and determine the effect on their progeny using the layered tissue co-culture system. Briefly, confluent human glioblastoma (T98G) cells received a single dose of 6 Gy or three daily doses of 2 Gy carbon ions, which were then seeded on top of an insert with bystander normal skin fibroblasts (NB1RGB) growing underneath. Cells were co-cultured for 6 h or allowed to grow for 20 population doublings, then harvested and assayed for different end points. A single dose of carbon ions resulted in less damage in bystander normal NB1RGB cells than the fractionated doses. In contrast, the progeny of bystander NB1RGB cells co-cultured with T98G cells exposed to fractionated doses showed less damage than progeny from bystander cells co-cultured with single dose glioblastoma cells. Furthermore, inhibition of gap junction communication demonstrated its involvement in the stressful effects in bystander cells and their progeny. These results indicate that dose fractionation reduced the late effect of carbon-ion exposure in the progeny of bystander cells compared to the effect in the initial bystander cells.

  17. Pro-apoptotic gene knockdown mediated by nanocomplexed siRNA reduces radiation damage in primary salivary gland cultures

    PubMed Central

    Arany, Szilvia; Xu, Qingfu; Hernady, Eric; Benoit, Danielle S.W.; Dewhurst, Steve; Ovitt, Catherine E.

    2012-01-01

    A critical issue in the management of head and neck tumors is radioprotection of the salivary glands. We have investigated whether siRNA-mediated gene knock down of pro-apoptotic mediators can reduce radiation-induced cellular apoptosis in salivary gland cells in vitro. We used novel, pH-responsive nanoparticles to deliver functionally active siRNAs into cultures of salivary gland cells. The nanoparticle molecules are comprised of cationic micelles that electrostatically interact with the siRNA, protecting it from nuclease attack, and also include pH-responsive endosomolytic constituents that promote release of the siRNA into the target cell cytoplasm. Transfection controls with Cy3-tagged siRNA/nanoparticle complexes showed efficiently internalized siRNAs in more than 70% of the submandibular gland cells. We found that introduction of siRNAs specifically targeting the Pkcδ or Bax genes significantly blocked the induction of these pro-apoptotic proteins that normally occurs after radiation in cultured salivary gland cells. Furthermore, the level of cell death from subsequent radiation, as measured by caspase-3, TUNEL, and mitochondrial disruption assays, was significantly decreased. Thus, we have successfully demonstrated that the siRNA/ nanoparticle-mediated knock down of pro-apoptotic genes can prevent radiation-induced damage in submandibular gland primary cell cultures. PMID:22253051

  18. Isolated Primary Blast Inhibits Long-Term Potentiation in Organotypic Hippocampal Slice Cultures.

    PubMed

    Vogel, Edward W; Effgen, Gwen B; Patel, Tapan P; Meaney, David F; Bass, Cameron R Dale; Morrison, Barclay

    2016-04-01

    Over the last 13 years, traumatic brain injury (TBI) has affected over 230,000 U.S. service members through the conflicts in Iraq and Afghanistan, mostly as a result of exposure to blast events. Blast-induced TBI (bTBI) is multi-phasic, with the penetrating and inertia-driven phases having been extensively studied. The effects of primary blast injury, caused by the shockwave interacting with the brain, remain unclear. Earlier in vivo studies in mice and rats have reported mixed results for primary blast effects on behavior and memory. Using a previously developed shock tube and in vitro sample receiver, we investigated the effect of isolated primary blast on the electrophysiological function of rat organotypic hippocampal slice cultures (OHSC). We found that pure primary blast exposure inhibited long-term potentiation (LTP), the electrophysiological correlate of memory, with a threshold between 9 and 39 kPa·ms impulse. This deficit occurred well below a previously identified threshold for cell death (184 kPa·ms), supporting our previously published finding that primary blast can cause changes in brain function in the absence of cell death. Other functional measures such as spontaneous activity, network synchronization, stimulus-response curves, and paired-pulse ratios (PPRs) were less affected by primary blast exposure, as compared with LTP. This is the first study to identify a tissue-level tolerance threshold for electrophysiological changes in neuronal function to isolated primary blast.

  19. Recent Developments in Understanding the Role of Aqueous Humor Outflow in Normal and Primary Open Angle Glaucoma

    PubMed Central

    Hann, Cheryl R.; Fautsch, Michael P.

    2015-01-01

    Primary open angle glaucoma (POAG) is the second leading cause of blindness in the world's rapidly aging population. POAG is characterized by progressive degeneration of neural structures in the posterior segment, often associated with a concomitant elevation of intraocular pressure. Changes in IOP are believed to be caused by a disruption in the normal outflow of aqueous humor. This article reviews recent research associated with normal and POAG aqueous humor outflow. Novel findings elucidating biochemical and pathological changes in the ocular tissues affected in POAG are presented. Stem cell research, identification of lymphatic markers, and increased use of mouse models give researchers exciting new tools to understand aqueous humor outflow, changes associated with POAG and identify underlying causes of the disease. PMID:26236568

  20. [The features of postsynaptic currents in primary culture of rat cortical neurons].

    PubMed

    Sibarov, D A; Antonov, S M

    2013-06-01

    The generation features of postsynaptic currents were studied in primary culture of cortical neurons at 7-20 days in vitro (DIV). The use of specific blockers of postsynaptic ion channels after 10 DIV revealed all types of electrical activity found in adult cortex including miniature inhibitory (mIPSCs), excitatory (mEPSCs) and spontaneous giant excitatory currents and spikes. The frequency of mEPSCs increased exponentially from 7 to 20 DIV doubling every 2.2 days in parallel with changes in action potentials generation. The mEPSCs generated by NMDA and AMPA or by only AMPA receptor activation were found. The inhibition of NMDA receptors by magnesium ions or AP5 were shown to modulate the frequency and amplitude of mEPSCs, which differ primary culture from brain slices possibly because of the lack of glial control of synaptic transmission.

  1. Expression profile of the matricellular protein osteopontin in primary open-angle glaucoma and the normal human eye.

    PubMed

    Chowdhury, Uttio Roy; Jea, Seung-Youn; Oh, Dong-Jin; Rhee, Douglas J; Fautsch, Michael P

    2011-08-16

    PURPOSE. To characterize the role of osteopontin (OPN) in primary open-angle glaucoma (POAG) and normal eyes. METHODS. OPN quantification was performed by enzyme-linked immunosorbent assay in aqueous humor (AH) obtained from human donor eyes (POAG and normal) and surgical samples (POAG and elective cataract removal). OPN expression and localization in whole eye tissue sections and primary normal human trabecular meshwork (NTM) cells were studied by Western blot and immunohistochemistry. Latanoprost-free acid (LFA)-treated NTM cells were analyzed for OPN gene and protein expression. Intraocular pressure was measured by tonometry, and central corneal thickness was measured by optical coherence tomography in young OPN(-/-) and wild-type mice. RESULTS. OPN levels were significantly reduced in donor POAG AH compared with normal AH (0.54 ± 0.18 ng/μg [n = 8] vs. 0.77 ± 0.23 ng/μg [n = 9]; P = 0.039). A similar trend was observed in surgical AH (1.05 ± 0.31 ng/μg [n = 20] vs. 1.43 ± 0.88 ng/μg [n = 20]; P = 0.083). OPN was present in the trabecular meshwork, corneal epithelium and endothelium, iris, ciliary body, retina, vitreous humor, and optic nerve. LFA increased OPN gene expression, but minimal change in OPN protein expression was observed. No difference in intraocular pressure (17.5 ± 2.0 mm Hg [n = 56] vs. 17.3 ± 1.9 mm Hg [n = 68]) but thinner central corneal thickness (91.7 ± 3.6 μm [n = 50] vs. 99.2 ± 5.5 μm [n = 70]) was noted between OPN(-/-) and wild-type mice. CONCLUSIONS. OPN is widely distributed in the human eye and was found in lower concentrations in POAG AH. Reduction of OPN in young mice does not affect IOP.

  2. Habitus and Flow in Primary School Musical Practice: Relations between Family Musical Cultural Capital, Optimal Experience and Music Participation

    ERIC Educational Resources Information Center

    Valenzuela, Rafael; Codina, Nuria

    2014-01-01

    Based on Bourdieu's idea that cultural capital is strongly related to family context, we describe the relations between family musical cultural capital and optimal experience during compulsory primary school musical practice. We analyse whether children from families with higher levels of musical cultural capital, and specifically with regard to…

  3. Habitus and Flow in Primary School Musical Practice: Relations between Family Musical Cultural Capital, Optimal Experience and Music Participation

    ERIC Educational Resources Information Center

    Valenzuela, Rafael; Codina, Nuria

    2014-01-01

    Based on Bourdieu's idea that cultural capital is strongly related to family context, we describe the relations between family musical cultural capital and optimal experience during compulsory primary school musical practice. We analyse whether children from families with higher levels of musical cultural capital, and specifically with regard to…

  4. Growth and antrum formation of bovine primary follicles in long-term culture in vitro.

    PubMed

    Sun, Jing; Li, Xiangdong

    2013-09-01

    Successful antral formation in vitro from bovine preantral follicles (145-170 μm) has been described previously, but antrum formation from the primary follicle (50-70 μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50-70 μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E2), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E2 + bFGF or FSH + LH + E2 + bFGF + EGF (p<0.05). An addition of 50 ng/ml bFGF or bFGF +25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF +25 ng/ml EGF to media containing FSH + LH + E2 turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.

  5. Blood culture contamination with Enterococci and skin organisms: implications for surveillance definitions of primary bloodstream infections.

    PubMed

    Freeman, Joshua T; Chen, Luke Francis; Sexton, Daniel J; Anderson, Deverick J

    2011-06-01

    Enterococci are a common cause of bacteremia but are also common contaminants. In our institution, approximately 17% of positive blood cultures with enterococci are mixed with skin organisms. Such isolates are probable contaminants. The specificity of the current definition of primary bloodstream infection could be increased by excluding enterococci mixed with skin organisms. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

  6. Human Immunodeficiency Virus Type 1 Coat Protein Neurotoxicity Mediated by Nitric Oxide in Primary Cortical Cultures

    NASA Astrophysics Data System (ADS)

    Dawson, Valina L.; Dawson, Ted M.; Uhl, George R.; Snyder, Solomon H.

    1993-04-01

    The human immunodeficiency virus type 1 coat protein, gp120, kills neurons in primary cortical cultures at low picomolar concentrations. The toxicity requires external glutamate and calcium and is blocked by glutamate receptor antagonists. Nitric oxide (NO) contributes to gp120 toxicity, since nitroarginine, an inhibitor of NO synthase, prevents toxicity as does deletion of arginine from the incubation medium and hemoglobin, which binds NO. Superoxide dismutase also attenuates toxicity, implying a role for superoxide anions.

  7. The Cultural Aspect of Foreign Languages Teaching at Primary School in Turkey

    ERIC Educational Resources Information Center

    Sadik, Turkoglu; Omer, Kocer

    2011-01-01

    Learning a foreign language is a new concept at primary school in Turkey. Even if the Minister of National Education has been thinking of it for a long time, it has only been compulsory since 2006. Pedagogues are still doing research on the way to teach a new language to children. One of the ideas is to base this new learning on cultural contents.…

  8. Characterisation and metabolism of astroglia-rich primary cultures from cathepsin K-deficient mice.

    PubMed

    Dauth, Stephanie; Schmidt, Maike M; Rehders, Maren; Dietz, Frank; Kelm, Sørge; Dringen, Ralf; Brix, Klaudia

    2012-09-01

    Cathepsin K is important for the brain, because its deficiency in mice is associated with a marked decrease in differentiated astrocytes and changes in neuronal patterning in the hippocampus as well as with learning and memory deficits. As cathepsin K activity is most prominent in hippocampal regions of wild type animals, we hypothesised alterations in astrocyte-mediated support of neurons as a potential mechanism underlying the impaired brain functions in cathepsin K-deficient mice. To address this hypothesis, we have generated and characterised astroglia-rich primary cell cultures from cathepsin K-deficient and wild type mice and compared these cultures for possible changes in metabolic support functions and cell composition. Interestingly, cells expressing the oligodendrocytic markers myelin-associated glycoprotein and myelin basic protein were more frequent in astroglia-rich cultures from cathepsin K-deficient mice. However, cell cultures from both genotypes were morphologically comparable and similar with respect to glucose metabolism. In addition, specific glutathione content, glutathione export and γ-glutamyl-transpeptidase activity remained unchanged, whereas the specific activities of glutathione reductase and glutathione-S-transferase were increased by around 50% in cathepsin K-deficient cultures. Thus, lack of cathepsin K in astroglia-rich cultures appears not to affect metabolic supply functions of astrocytes but to facilitate the maturation of oligodendrocytes.

  9. Are primary healthcare services culturally appropriate for Aboriginal people? Findings from a remote community.

    PubMed

    Smith, Kaye; Fatima, Yaqoot; Knight, Sabina

    2017-04-13

    This study explored the views of key stakeholders on cultural appropriateness of primary health care (PHC) services for Aboriginal people. A total of 78 participants, including healthcare providers, administrative team members (n=24, ~30% of study sample) and Aboriginal community members (n=54, ~70% of study sample) living in remote North West Queensland participated in the study. Outcome measures were assessed by administering survey questionnaires comprising qualitative questions and various subscales (e.g. provider behaviours and attitudes, communication, physical environment and facilities, and support from administrative staff). Descriptive statistics were used to present quantitative findings, whereas inductive thematic analysis was used for qualitative data. In contrast to the views of PHC providers, a significant number of Aboriginal people did not perceive that they were receiving culturally appropriate services. Although PHC providers acknowledged cultural awareness training for familiarising themselves with Aboriginal culture, they found the training to be general, superficial and lacking prospective evaluation. PHC providers should understand that culturally inappropriate clinical encounters generate mistrust and dissatisfaction. Therefore, a broad approach involving culturally respectful association between PHC providers, Aboriginal consumers and administrative staff is required to bring sustainable changes at the practice level to improve the health of Aboriginal people.

  10. Multiple follicle culture supports primary follicle growth through paracrine-acting signals.

    PubMed

    Hornick, J E; Duncan, F E; Shea, L D; Woodruff, T K

    2013-01-01

    In vitro follicle growth in alginate hydrogels is a unique and versatile method for studying ovarian and follicle biology that may also have implications for fertility preservation. Current culture systems support the development of isolated mouse follicles from the secondary stage onward. However, it has been a challenge to grow smaller follicles in vitro due to the dissociation of the oocyte from companion somatic cells. Recent work has demonstrated that coculturing primary follicles with mouse embryonic fibroblasts or ovarian stromal cells supports follicle survival and growth. In this study, we demonstrate that follicles themselves can exert a beneficial coculture effect. When primary follicles were cultured in groups of five or ten (multiple follicle culture), there was increased growth and survival. The multiple follicle culture approach maintained follicle integrity and resulted in the formation of antral stage follicles containing meiotically competent gametes. The growth and survival of primary follicles were highly number dependent, with the most significant enhancement observed when the largest number of follicles was grown together. Our data suggest that the follicle unit is necessary to produce the secreted factors responsible for the supportive effects of multiple follicle culture, as neither denuded oocytes, oocyte-secreted factors, nor granulosa cells alone were sufficient to support early follicle growth in vitro. Therefore, there may be signaling from both the oocyte and the follicle that enhances growth but requires both components in a feedback mechanism. This work is consistent with current in vivo models for follicle growth and thus advances the movement to recapitulate the ovarian environment in vitro.

  11. Characterization of abalone Haliotis tuberculata-Vibrio harveyi interactions in gill primary cultures.

    PubMed

    Pichon, Delphine; Cudennec, Benoit; Huchette, Sylvain; Djediat, Chakib; Renault, Tristan; Paillard, Christine; Auzoux-Bordenave, Stéphanie

    2013-10-01

    The decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell-V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a non-pathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy. During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments.

  12. Quantification and Characterization of UVB-Induced Mitochondrial Fragmentation in Normal Primary Human Keratinocytes

    PubMed Central

    Jugé, Romain; Breugnot, Josselin; Da Silva, Célia; Bordes, Sylvie; Closs, Brigitte; Aouacheria, Abdel

    2016-01-01

    UV irradiation is a major environmental factor causing skin dryness, aging and cancer. UVB in particular triggers cumulative DNA damage, oxidative stress and mitochondrial dysfunction. The objective of our study was to provide both qualitative and quantitative analysis of how mitochondria respond to UVB irradiation in normal human epidermal keratinocytes (NHEK) of healthy donors, with the rationale that monitoring mitochondrial shape will give an indication of cell population fitness and enable the screening of bioactive agents with UVB-protective properties. Our results show that NHEK undergo dose-dependent mitochondrial fragmentation after exposure to UVB. In order to obtain a quantitative measure of this phenomenon, we implemented a novel tool for automated quantification of mitochondrial morphology in live cells based on confocal microscopy and computational calculations of mitochondrial shape descriptors. This method was used to substantiate the effects on mitochondrial morphology of UVB irradiation and of knocking-down the mitochondrial fission-mediating GTPase Dynamin-related protein 1 (DRP1). Our data further indicate that all the major mitochondrial dynamic proteins are expressed in NHEK but that their level changes were stronger after mitochondrial uncoupler treatment than following UVB irradiation or DRP1 knock-down. Our system and procedures might be of interest for the identification of cosmetic or dermatologic UVB-protective agents. PMID:27731355

  13. Differences in the Tongue Features of Primary Dysmenorrhea Patients and Controls over a Normal Menstrual Cycle

    PubMed Central

    Lee, Haebeom

    2017-01-01

    Background The aims of this study were to investigate the relationships between tongue features and the existence of menstrual pain and to provide basic information regarding the changes in tongue features during a menstrual cycle. Methods This study was conducted at the Kyung Hee University Medical Center. Forty-eight eligible participants aged 20 to 29 years were enrolled and assigned to two groups according to their visual analogue scale (VAS) scores. Group A included 24 females suffering from primary dysmenorrhea (PD) caused by qi stagnation and blood stasis syndrome with VAS ≥ 4. In contrast, Group B included 24 females with few premenstrual symptoms and VAS < 4. All participants completed four visits (menses-follicular-luteal-menses phases), and the tongue images were taken by using a computerized tongue image analysis system (CTIS). Results The results revealed that the tongue coating color value and the tongue coating thickness in the PD group during the menstrual phase were significantly lower than those of the control group (P = 0.031 and P = 0.029, resp.). Conclusions These results suggest that the tongue features obtained from the CTIS may serve as a supplementary means for the differentiation of syndromes and the evaluation of therapeutic effect and prognosis in PD. Trial Registration This trial was registered with Clinical Research Information Service, registration number KCT0001604, registered on 27 August 2015. PMID:28642801

  14. A hybrid substratum for primary hepatocyte culture that enhances hepatic functionality with low serum dependency.

    PubMed

    Meng, Qingyuan; Tao, Chunsheng; Qiu, Zhiye; Akaike, Toshihiro; Cui, Fuzhai; Wang, Xiumei

    2015-01-01

    Cell culture systems have proven to be crucial for the in vitro maintenance of primary hepatocytes and the preservation of hepatic functional expression at a high level. A poly-(N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide) matrix can recognize cells and promote liver function in a spheroid structure because of a specific galactose-asialoglycoprotein receptor interaction. Meanwhile, a fusion protein, E-cadherin-Fc, when incubated with various cells, has shown an enhancing effect on cellular viability and metabolism. Therefore, a hybrid substratum was developed for biomedical applications by using both of these materials to combine their advantages for primary hepatocyte cultures. The isolated cells showed a monolayer aggregate morphology on the coimmobilized surface and displayed higher functional expression than cells on traditional matrices. Furthermore, the hybrid system, in which the highest levels of cell adhesion and hepatocellular metabolism were achieved with the addition of 1% fetal bovine serum, showed a lower serum dependency than the collagen/gelatin-coated surface. Accordingly, this substrate may attenuate the negative effects of serum and further contribute to establishing a defined culture system for primary hepatocytes.

  15. Polystyrene-coated micropallets for culture and separation of primary muscle cells

    PubMed Central

    Detwiler, David A.; Dobes, Nicholas C.; Sims, Christopher E.; Kornegay, Joseph N.; Allbritton, Nancy L.

    2011-01-01

    Despite identification of a large number of adult stem cell types, current primary cell isolation and identification techniques yield heterogeneous samples, making detailed biological studies challenging. To identify subsets of isolated cells, technologies capable of simultaneous cell culture and cloning are necessary. Micropallet arrays, a new cloning platform for adherent cell types, hold great potential. However, the microstructures composing these arrays are fabricated from an epoxy photoresist 1002F, a growth surface unsuitable for many cell types. Optimization of the microstructures’ surface properties was conducted for the culture of satellite cells, primary muscle cells for which improved cell isolation techniques are desired. A variety of surface materials were screened for satellite cell adhesion and proliferation and compared to their optimal substrate, gelatin-coated Petri dishes. A 1-μm thick, polystyrene copolymer was applied to the microstructures by contact-printing. A negatively charged copolymer of 5% acrylic acid in 95% styrene was found to be equivalent to the control Petri dishes for cell adhesion and proliferation. Cells cultured on control dishes and optimal copolymer-coated surfaces maintained an undifferentiated state and showed similar mRNA expression for two genes indicative of cell differentiation during a standard differentiation protocol. Experiments using additional contact-printed layers of extracellular matrix proteins collagen and gelatin showed no further improvements. This micropallet coating strategy is readily adaptable to optimize the array surface for other types of primary cells. PMID:22159513

  16. Effects of the polymeric niche on neural stem cell characteristics during primary culturing.

    PubMed

    Haubenwallner, Stefan; Katschnig, Matthias; Fasching, Ulrike; Patz, Silke; Trattnig, Christa; Andraschek, Natascha; Grünbacher, Gerda; Absenger, Markus; Laske, Stephan; Holzer, Clemens; Balika, Werner; Wagner, Manuela; Schäfer, Ute

    2014-05-01

    The polymeric niche encountered by cells during primary culturing can affect cell fate. However, most cell types are primarily propagated on polystyrene (PS). A cell type specific screening for optimal primary culture polymers particularly for regenerative approaches seems inevitable. The effect of physical and chemical properties of treated (corona, oxygen/nitrogen plasma) and untreated cyclic olefin polymer (COP), polymethymethacrylate (PMMA), PP, PLA, PS, PC on neuronal stem cell characteristics was analyzed. Our comprehensive approach revealed plasma treated COP and PMMA as optimal polymers for primary neuronal stem cell culturing and propagation. An increase in the number of NT2/D1 cells with pronounced adhesion, metabolic activities and augmented expression of neural precursor markers was associated to the plasma treatment of surfaces of COP and PMMA with nitrogen or oxygen, respectively. A shift towards large cell sizes at stable surface area/volume ratios that might promote the observed increase in metabolic activities and distinct modulations in F-actin arrangements seem to be primarily mediated by the plasma treatment of surfaces. These results indicate that the polymeric niche has a distinct impact on various cell characteristics. The selection of distinct polymers and the controlled design of an optimized polymer microenvironment might thereby be an effective tool to promote essential cell characteristics for subsequent approaches.

  17. Diabetes increases susceptibility of primary cultures of rat proximal tubular cells to chemically induced injury

    SciTech Connect

    Zhong Qing; Terlecky, Stanley R.; Lash, Lawrence H.

    2009-11-15

    Diabetic nephropathy is characterized by increased oxidative stress and mitochondrial dysfunction. In the present study, we prepared primary cultures of proximal tubular (PT) cells from diabetic rats 30 days after an ip injection of streptozotocin and compared their susceptibility to oxidants (tert-butyl hydroperoxide, methyl vinyl ketone) and a mitochondrial toxicant (antimycin A) with that of PT cells isolated from age-matched control rats, to test the hypothesis that PT cells from diabetic rats exhibit more cellular and mitochondrial injury than those from control rats when exposed to these toxicants. PT cells from diabetic rats exhibited higher basal levels of reactive oxygen species (ROS) and higher mitochondrial membrane potential, demonstrating that the PT cells maintain the diabetic phenotype in primary culture. Incubation with either the oxidants or mitochondrial toxicant resulted in greater necrotic and apoptotic cell death, greater evidence of morphological damage, greater increases in ROS, and greater decreases in mitochondrial membrane potential in PT cells from diabetic rats than in those from control rats. Pretreatment with either the antioxidant N-acetyl-L-cysteine or a catalase mimetic provided equivalent protection of PT cells from both diabetic and control rats. Despite the greater susceptibility to oxidative and mitochondrial injury, both cytoplasmic and mitochondrial glutathione concentrations were markedly higher in PT cells from diabetic rats, suggesting an upregulation of antioxidant processes in diabetic kidney. These results support the hypothesis that primary cultures of PT cells from diabetic rats are a valid model in which to study renal cellular function in the diabetic state.

  18. Toxic effects of anabolic-androgenic steroids in primary rat hepatic cell cultures.

    PubMed

    Welder, A A; Robertson, J W; Melchert, R B

    1995-08-01

    Hepatic complications in athletes and bodybuilders after abusing anabolic-androgenic steroids (AAS) have been reported. Hepatic injury, including cholestasis, peliosis hepatis, hyperplasia, and tumors, have been attributed to abuse of the 17 alpha-alkylated AAS. Some of these pathological conditions have been reversed when individuals were converted to nonalkylated AAS regimens. The purpose of this study was to determine and compare the direct toxic effects of commonly abused AAS (both 17 alpha-alkylated and nonalkylated) in primary hepatic cell cultures. Primary cultures, established from 60-day-old Sprague-Dawley rats, were exposed to doses of 1 x 10(-8), 1 x 10(-6), and 1 x 10(-4)M 19-nortestosterone, fluoxymesterone, testosterone cypionate, stanozolol, danazol, oxymetholone, testosterone, estradiol, and methyltestosterone for 1, 4, and 24 hr. Lactate dehydrogenase (LDH) release, neutral red (NR) retention, and glutathione (GSH) depletion were evaluated to determine plasma membrane damage, cell viability, and possible oxidative injury, respectively. Those cultures exposed to the 17 alpha-alkylated AAS, methyltestosterone and stanozolol, at doses of 1 x 10(-4) M for 24 hr and the 17 alpha-alkylated AAS, oxymetholone, at 1 x 10(-4) M for 4 and 24 hr showed significant increased in LDH release and decreases in NR retention while there were no significant differences with the nonalkylated steroids (testosterone cypionate, 19-nortestosterone, testosterone, and estradiol). GSH depletion was evaluated in cultures treated with 1 x 10(-8), 1 x 10(-6), and 1 x 10(-4) M concentrations of methyltestosterone, stanozolol, and oxymetholone for 1, 2, 4, and 6 hr. Cultures exposed to 1 x 10(-4) M oxymetholone were significantly depleted of GSH at 2, 4, and 6 hr; cultures exposed to 1 x 10(-4) M methyltestosterone were significantly depleted of GSH at 4 and 6 hr; and cultures exposed to stanozolol were not significantly depleted of GSH at any of the time periods tested. These

  19. Primary Retinal Cultures as a Tool for Modeling Diabetic Retinopathy: An Overview

    PubMed Central

    Varano, Monica; Mallozzi, Cinzia; Gaddini, Lucia; Formisano, Giuseppe; Pricci, Flavia

    2015-01-01

    Experimental models of diabetic retinopathy (DR) have had a crucial role in the comprehension of the pathophysiology of the disease and the identification of new therapeutic strategies. Most of these studies have been conducted in vivo, in animal models. However, a significant contribution has also been provided by studies on retinal cultures, especially regarding the effects of the potentially toxic components of the diabetic milieu on retinal cell homeostasis, the characterization of the mechanisms on the basis of retinal damage, and the identification of potentially protective molecules. In this review, we highlight the contribution given by primary retinal cultures to the study of DR, focusing on early neuroglial impairment. We also speculate on possible themes into which studies based on retinal cell cultures could provide deeper insight. PMID:25688355

  20. Harvesting Human Prostate Tissue Material and Culturing Primary Prostate Epithelial Cells.

    PubMed

    Frame, Fiona M; Pellacani, Davide; Collins, Anne T; Maitland, Norman J

    2016-01-01

    In order to fully explore the biology of a complex solid tumor such as prostate cancer, it is desirable to work with patient tissue. Only by working with cells from a tissue can we take into account patient variability and tumor heterogeneity. Cell lines have long been regarded as the workhorse of cancer research and it could be argued that they are of most use when considered within a panel of cell lines, thus taking into account specified mutations and variations in phenotype between different cell lines. However, often very different results are obtained when comparing cell lines to primary cells cultured from tissue. It stands to reason that cells cultured from patient tissue represents a close-to-patient model that should and does produce clinically relevant data. This chapter aims to illustrate the methods of processing, storing and culturing cells from prostate tissue, with a description of potential uses.

  1. Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration.

    PubMed

    Radad, Khaled; Rausch, Wolf-Dieter; Gille, Gabriele

    2006-09-01

    Although the definite etiology of Parkinson's disease is still unclear, increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in increasing the risk of developing Parkinson's disease. In the present study, primary cultures prepared from embryonic mouse mesencephala were applied to investigate the toxic effects and underlying mechanisms of rotenone-induced neuronal cell death relevant to Parkinson's disease. Results revealed that rotenone destroyed dopaminergic neurons in a dose- and time-dependent manner. Consistent with the cytotoxic effect of rotenone as evidenced by dopaminergic cell loss, it significantly increased the release of lactate dehydrogenase into the culture medium, the number of necrotic cells in the culture and the number of nuclei showing apoptotic features. Rotenone exerted toxicity by decreasing the mitochondrial membrane potential, increasing reactive oxygen species production and shifting respiration to a more anaerobic state.

  2. Aragonite crystallization in primary cell cultures of multicellular isolates from a hard coral, Pocillopora damicornis.

    PubMed

    Domart-Coulon, I J; Elbert, D C; Scully, E P; Calimlim, P S; Ostrander, G K

    2001-10-09

    The foundation of marine coral reef ecosystems is calcium carbonate accumulated primarily by the action of hard corals (Coelenterata: Anthozoa: Scleractinia). Colonial hard coral polyps cover the surface of the reef and deposit calcium carbonate as the aragonite polymorph, stabilized into a continuous calcareous skeleton. Scleractinian coral skeleton composition and architecture are well documented; however, the cellular mechanisms of calcification are poorly understood. There is little information on the nature of the coral cell types involved or their cooperation in biocalcification. We report aragonite crystallization in primary cell cultures of a hard coral, Pocillopora damicornis. Cells of apical coral colony fragments were isolated by spontaneous in vitro dissociation. Single dissociated cell types were separated by density in a discontinuous Percoll gradient. Primary cell cultures displayed a transient increase in alkaline phosphatase (ALP) activity, to the level observed in intact corals. In adherent multicellular isolate cultures, enzyme activation was followed by precipitation of aragonite. Modification of the ionic formulation of the medium prolonged maintenance of isolates, delayed ALP activation, and delayed aragonite precipitation. These results demonstrate that in vitro crystallization of aragonite in coral cell cultures is possible, and provides an innovative approach to investigate reef-building coral calcification at the cellular level.

  3. Photodynamic therapy (PDT) of endometrium primary cultures serving as an in-vitro model for endometriosis

    NASA Astrophysics Data System (ADS)

    Herter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

    1994-05-01

    As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

  4. Photodynamic treatment (PDT) of endometrium primary cultures serving as an in-vitro-model for endometriosis

    NASA Astrophysics Data System (ADS)

    Werter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

    1994-05-01

    As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

  5. Aragonite crystallization in primary cell cultures of multicellular isolates from a hard coral, Pocillopora damicornis

    PubMed Central

    Domart-Coulon, Isabelle J.; Elbert, David C.; Scully, Erik P.; Calimlim, Precilia S.; Ostrander, Gary K.

    2001-01-01

    The foundation of marine coral reef ecosystems is calcium carbonate accumulated primarily by the action of hard corals (Coelenterata: Anthozoa: Scleractinia). Colonial hard coral polyps cover the surface of the reef and deposit calcium carbonate as the aragonite polymorph, stabilized into a continuous calcareous skeleton. Scleractinian coral skeleton composition and architecture are well documented; however, the cellular mechanisms of calcification are poorly understood. There is little information on the nature of the coral cell types involved or their cooperation in biocalcification. We report aragonite crystallization in primary cell cultures of a hard coral, Pocillopora damicornis. Cells of apical coral colony fragments were isolated by spontaneous in vitro dissociation. Single dissociated cell types were separated by density in a discontinuous Percoll gradient. Primary cell cultures displayed a transient increase in alkaline phosphatase (ALP) activity, to the level observed in intact corals. In adherent multicellular isolate cultures, enzyme activation was followed by precipitation of aragonite. Modification of the ionic formulation of the medium prolonged maintenance of isolates, delayed ALP activation, and delayed aragonite precipitation. These results demonstrate that in vitro crystallization of aragonite in coral cell cultures is possible, and provides an innovative approach to investigate reef-building coral calcification at the cellular level. PMID:11593000

  6. Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections

    PubMed Central

    Rajaram, Anuradha; Kotrashetti, Vijayalakshmi S.; Somannavar, Pradeep D.; Ingalagi, Preeti; Bhat, Kishore

    2016-01-01

    Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion. The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections. PMID:27651878

  7. Technical aspects of oxygen level regulation in primary cell cultures: A review

    PubMed Central

    2016-01-01

    Oxygen (O2) is an essential element for aerobic respiration. Atmospheric concentration of O2 is approximately 21%. Mammalian cells, however, are generally adapted to O2 levels much lower than atmospheric conditions. The pericellular levels of O2 must also be maintained within a fairly narrow range to meet the demands of cells. This applies equally to cells in vivo and cells in primary cultures. There has been growing interest in the performance of cell culture experiments under various O2 levels to study molecular and cellular responses. To this end, a range of technologies (e.g. gas-permeable technology) and instruments (e.g. gas-tight boxes and gas-controlled incubators) have been developed. It should be noted, however, that some of these have limitations and they are still undergoing refinement. Nevertheless, better results should be possible when technical concerns are taken into account. This paper aims to review various aspects of O2 level adjustment in primary cell cultures, regulation of pericellular O2 gradients and possible effects of the cell culture medium. PMID:28652851

  8. Protective Effect of Edaravone against Carbon Monoxide Induced Apoptosis in Rat Primary Cultured Astrocytes

    PubMed Central

    Xu, Xiaodan; Zhang, Hong; Wang, Ke; Tu, Tao

    2017-01-01

    Objective. To observe the protective effect of edaravone (Eda) on astrocytes after prolonged exposure to carbon monoxide (CO) and further to investigate the potential mechanisms of Eda against CO-induced apoptosis. Methods. The rat primary cultured astrocytes were cultured in vitro and exposed to 1% CO for 24 h after being cultured with different concentrations of Eda. MTT assay was used to detect the cytotoxicity of CO. Flow cytometry was used to detect the apoptosis rate, membrane potential of mitochondria, and ROS level. The mRNA and protein expressions of Bcl-2, Bax, and caspase-3 were assessed by real-time PCR and Western blotting analysis, respectively. Results. Eda can significantly suppress cytotoxicity of CO, and it can significantly increase membrane potential of mitochondria and Bcl-2 expressions and significantly suppress the apoptosis rate, ROS level, Bax, and caspase-3 expressions. Conclusion. Eda protects against CO-induced apoptosis in rat primary cultured astrocytes through decreasing ROS production and subsequently inhibiting mitochondrial apoptosis pathway. PMID:28261501

  9. E-Cigarette Affects the Metabolome of Primary Normal Human Bronchial Epithelial Cells.

    PubMed

    Aug, Argo; Altraja, Siiri; Kilk, Kalle; Porosk, Rando; Soomets, Ursel; Altraja, Alan

    2015-01-01

    E-cigarettes are widely believed to be safer than conventional cigarettes and have been even suggested as aids for smoking cessation. However, while reasonable with some regards, this judgment is not yet supported by adequate biomedical research data. Since bronchial epithelial cells are the immediate target of inhaled toxicants, we hypothesized that exposure to e-cigarettes may affect the metabolome of human bronchial epithelial cells (HBEC) and that the changes are, at least in part, induced by oxidant-driven mechanisms. Therefore, we evaluated the effect of e-cigarette liquid (ECL) on the metabolome of HBEC and examined the potency of antioxidants to protect the cells. We assessed the changes of the intracellular metabolome upon treatment with ECL in comparison of the effect of cigarette smoke condensate (CSC) with mass spectrometry and principal component analysis on air-liquid interface model of normal HBEC. Thereafter, we evaluated the capability of the novel antioxidant tetrapeptide O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1) to attenuate the effect of ECL. ECL caused a significant shift in the metabolome that gradually gained its maximum by the 5th hour and receded by the 7th hour. A second alteration followed at the 13th hour. Treatment with CSC caused a significant initial shift already by the 1st hour. ECL, but not CSC, significantly increased the concentrations of arginine, histidine, and xanthine. ECL, in parallel with CSC, increased the content of adenosine diphosphate and decreased that of three lipid species from the phosphatidylcholine family. UPF1 partially counteracted the ECL-induced deviations, UPF1's maximum effect occurred at the 5th hour. The data support our hypothesis that ECL profoundly alters the metabolome of HBEC in a manner, which is comparable and partially overlapping with the effect of CSC. Hence, our results do not support the concept of harmlessness of e-cigarettes.

  10. Senescence Process in Primary Wilms' Tumor Cell Culture Induced by p53 Independent p21 Expression.

    PubMed

    Theerakitthanakul, Korkiat; Khrueathong, Jeerasak; Kruatong, Jirasak; Graidist, Potchanapond; Raungrut, Pritsana; Kayasut, Kanita; Sangkhathat, Surasak

    2016-01-01

    Wilms tumor (WT) is an embryonal tumor occurring in developing kidney tissue. WT cells showing invasive cancer characteristics, also retain renal stem cell behaviours. In-vitro culture of WT is hampered by limited replicative potential. This study aimed to establish a longterm culture of WT cells to enable the study of molecular events to attempt to explain its cellular senescence. Primary cell cultures from fresh WT tumor specimen were established. Of 5 cultures tried, only 1 could be propagated for more than 7 passages. One culture, identified as PSU-SK-1, could be maintained > 35 passages and was then subjected to molecular characterization and evaluation for cancer characteristics. The cells consistently harbored concomitant mutations of CTNNB1 (Ser45Pro) and WT1 (Arg413Stop) thorough the cultivation. On Transwell invasion assays, the cells exhibited migration and invasion at 55% and 27% capability of the lung cancer cells, A549. On gelatin zymography, PSU-SK-1 showed high expression of the matrix metaloproteinase. The cells exhibited continuous proliferation with 24-hour doubling time until passages 28-30 when the growth slowed, showing increased cell size, retention of cells in G1/S proportion and positive β-galactosidase staining. As with those evidence of senescence in advanced cell passages, expression of p21 and cyclin D1 increased when the expression of β-catenin and its downstream protein, TCF, declined. There was also loss-of-expression of p53 in this cell line. In conclusion, cellular senescence was responsible for limited proliferation in the primary culture of WT, which was also associated with increased expression of p21 and was independent of p53 expression. Decreased activation of the Wnt signalling might explain the induction of p21 expression.

  11. Senescence Process in Primary Wilms' Tumor Cell Culture Induced by p53 Independent p21 Expression

    PubMed Central

    Theerakitthanakul, Korkiat; Saetang, Jirakrit; Kruatong, Jirasak; Graidist, Potchanapond; Raungrut, Pritsana; Kayasut, Kanita; Sangkhathat, Surasak

    2016-01-01

    Wilms tumor (WT) is an embryonal tumor occurring in developing kidney tissue. WT cells showing invasive cancer characteristics, also retain renal stem cell behaviours. In-vitro culture of WT is hampered by limited replicative potential. This study aimed to establish a longterm culture of WT cells to enable the study of molecular events to attempt to explain its cellular senescence. Methods: Primary cell cultures from fresh WT tumor specimen were established. Of 5 cultures tried, only 1 could be propagated for more than 7 passages. One culture, identified as PSU-SK-1, could be maintained > 35 passages and was then subjected to molecular characterization and evaluation for cancer characteristics. The cells consistently harbored concomitant mutations of CTNNB1 (Ser45Pro) and WT1 (Arg413Stop) thorough the cultivation. On Transwell invasion assays, the cells exhibited migration and invasion at 55% and 27% capability of the lung cancer cells, A549. On gelatin zymography, PSU-SK-1 showed high expression of the matrix metaloproteinase. The cells exhibited continuous proliferation with 24-hour doubling time until passages 28-30 when the growth slowed, showing increased cell size, retention of cells in G1/S proportion and positive β-galactosidase staining. As with those evidence of senescence in advanced cell passages, expression of p21 and cyclin D1 increased when the expression of β-catenin and its downstream protein, TCF, declined. There was also loss-of-expression of p53 in this cell line. In conclusion, cellular senescence was responsible for limited proliferation in the primary culture of WT, which was also associated with increased expression of p21 and was independent of p53 expression. Decreased activation of the Wnt signalling might explain the induction of p21 expression. PMID:27698927

  12. Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa

    SciTech Connect

    Chew, C.S.; Ljungstroem, M.S.; Smolka, A.; Brown, M.R.

    1989-01-01

    A new procedure for isolation and primary culture of gastric parietal cells is described. Parietal cells from rabbit gastric mucosa are enriched to greater than 95% purity by combining a Nycodenz gradient separation with centrifugal elutriation. Cells are plated on the basement membrane matrix, Matrigel, and maintained in culture for at least 1 wk. Parietal cells cultured in this manner remain differentiated, cross-react with monoclonal H+-K+-ATPase antibodies, and respond to histamine, gastrin, and cholinergic stimulation with increased acid production as measured by accumulation of the weak base, (/sup 14/C)aminopyrine. When stimulated, cultured cells undergo ultrastructural changes in which intracellular canaliculi expand and numerous microvilli are observed. These ultrastructural changes are similar to those previously found to occur in vivo and in acutely isolated parietal cells. Morphological transformations in living cells can also be observed with differential interference contrast optics in the light microscope. After histamine stimulation, intracellular canaliculi gradually expand to form large vacuolar spaces. When the H2 receptor antagonist, cimetidine, is added to histamine-stimulated cells, these vacuoles gradually disappear. The ability to maintain hormonally responsive parietal cells in primary culture should make it possible to study direct, long-term effects of a variety of agonists and antagonists on parietal cell secretory-related activity. These cultured cells should also prove to be useful for the study of calcium transients, ion fluxes, and intracellular pH as related to acid secretion in single cells, particularly since morphological transformations can be used to monitor physiological responses at the same time within the same cell.

  13. Primary cultures of human colon cancer as a model to study cancer stem cells.

    PubMed

    Koshkin, Sergey; Danilova, Anna; Raskin, Grigory; Petrov, Nikolai; Bajenova, Olga; O'Brien, Stephen J; Tomilin, Alexey; Tolkunova, Elena

    2016-09-01

    The principal cause of death in cancer involves tumor progression and metastasis. Since only a small proportion of the primary tumor cells, cancer stem cells (CSCs), which are the most aggressive, have the capacity to metastasize and display properties of stem cells, it is imperative to characterize the gene expression of diagnostic markers and to evaluate the drug sensitivity in the CSCs themselves. Here, we have examined the key genes that are involved in the progression of colorectal cancer and are expressed in cancer stem cells. Primary cultures of colorectal cancer cells from a patient's tumors were studied using the flow cytometry and cytological methods. We have evaluated the clinical and stem cell marker expression in these cells, their resistance to 5-fluorouracil and irinotecan, and the ability of cells to form tumors in mice. The data shows the role of stem cell marker Oct4 in the resistance of primary colorectal cancer tumor cells to 5-fluorouracil.

  14. Excitability of the Primary Motor Cortex Increases More Strongly with Slow- than with Normal-Speed Presentation of Actions

    PubMed Central

    Moriuchi, Takefumi; Iso, Naoki; Sagari, Akira; Ogahara, Kakuya; Kitajima, Eiji; Tanaka, Koji; Tabira, Takayuki; Higashi, Toshio

    2014-01-01

    Introduction The aim of the present study was to investigate how the speed of observed action affects the excitability of the primary motor cortex (M1), as assessed by the size of motor evoked potentials (MEPs) induced by transcranial magnetic stimulation (TMS). Methods Eighteen healthy subjects watched a video clip of a person catching a ball, played at three different speeds (normal-, half-, and quarter-speed). MEPs were induced by TMS when the model's hand had opened to the widest extent just before catching the ball (“open”) and when the model had just caught the ball (“catch”). These two events were locked to specific frames of the video clip (“phases”), rather than occurring at specific absolute times, so that they could easily be compared across different speeds. MEPs were recorded from the thenar (TH) and abductor digiti minimi (ADM) muscles of the right hand. Results The MEP amplitudes were higher when the subjects watched the video clip at low speed than when they watched the clip at normal speed. A repeated-measures ANOVA, with the factor VIDEO-SPEED, showed significant main effects. Bonferroni's post hoc test showed that the following MEP amplitude differences were significant: TH, normal vs. quarter; ADM, normal vs. half; and ADM, normal vs. quarter. Paired t-tests showed that the significant MEP amplitude differences between TMS phases under each speed condition were TH, “catch” higher than “open” at quarter speed; ADM, “catch” higher than “open” at half speed. Conclusions These results indicate that the excitability of M1 was higher when the observed action was played at low speed. Our findings suggest that the action observation system became more active when the subjects observed the video clip at low speed, because the subjects could then recognize the elements of action and intention in others. PMID:25479161

  15. Overweight and obese patients in a primary care population report less sleep than patients with a normal body mass index.

    PubMed

    Vorona, Robert D; Winn, Maria P; Babineau, Teresa W; Eng, Benjamin P; Feldman, Howard R; Ware, J Catesby

    2005-01-10

    Insufficient sleep and obesity are common in the United States. Restricted sleep causes important neurocognitive changes, including excessive daytime sleepiness and altered mood. This may result in work-related injuries and automotive crashes. Evidence links sleep loss to hormonal changes that could result in obesity. This article examines the association between restricted sleep and obesity in a heterogeneous adult primary care population. A total of 1001 patients from 4 primary care practices participated in this prospective study. Patients completed a questionnaire administered by a nurse or study coordinator concerning demographics, medical problems, sleep habits, and sleep disorders. Professional staff measured height and weight in the office. The relationship between body mass index (BMI) and reported total sleep time per 24 hours was analyzed after categorizing patients according to their BMI (calculated as weight in kilograms divided by the square of height in meters) as being of normal weight (<25), overweight (25-29.9), obese (30-39.9), or extremely obese (> or =40). Analyzable forms from 924 patients aged between 18 and 91 years indicated that (1) the mean BMI was 30; (2) women slept more than men; (3) overweight and obese patients slept less than patients with a normal BMI (patients reported less sleep in a nearly linear relationship from the normal through the obese group); and (4) this trend of decreasing sleep time was reversed in the extremely obese patients. This study found that reduced amounts of sleep are associated with overweight and obese status. Interventions manipulating total sleep time could elucidate a cause-and-effect relationship between insufficient sleep and obesity.

  16. Epithelial-mesenchymal transitions during cell culture of primary thyroid tumors?

    PubMed

    Herrmann, M E; Trevor, K T

    1993-04-01

    Fibroblast contamination of epithelial tumor cell cultures is of great concern when examining tumor cells in vitro for specific biochemical and cytogenetic changes. The observations of normal karyotypes in thyroid tumor cell cultures have raised the concern of whether residual tissue fibroblasts might obscure the cytogenetic analysis of transformed epithelial cells. We have characterized early passaged thyroid tumor cells to examine the proportions of epithelial and fibroblastic cell types. Cells were analyzed by immunocytology using antibodies recognizing the thyroid prohormone thyroglobulin, epithelial cytokeratins, and vimentin, a mesenchyme marker. Tumors consisted of one follicular adenoma and five papillary carcinomas. When examined by day 15 in culture, all cells contained filaments composed of vimentin, which most likely represents an adaptation to culture conditions. Double immunofluorescence staining for thyroglobulin and cytokeratin revealed the presence of not only epithelial but also spindle-like fibroblastoid cells possessing thyroid epithelial cell markers. The results suggest that in thyroid tumor cultures there is a unique cell type intermediate between epithelial and mesenchyme phenotypes that must be considered when performing cytogenetic analysis.

  17. Primary liver cells cultured on carbon nanotube substrates for liver tissue engineering and drug discovery applications.

    PubMed

    Che Abdullah, Che Azurahanim; Azad, Chihye Lewis; Ovalle-Robles, Raquel; Fang, Shaoli; Lima, Marcio D; Lepró, Xavier; Collins, Steve; Baughman, Ray H; Dalton, Alan B; Plant, Nick J; Sear, Richard P

    2014-07-09

    Here, we explore the use of two- and three-dimensional scaffolds of multiwalled-carbon nanotubes (MWNTs) for hepatocyte cell culture. Our objective is to study the use of these scaffolds in liver tissue engineering and drug discovery. In our experiments, primary rat hepatocytes, the parenchymal (main functional) cell type in the liver, were cultured on aligned nanogrooved MWNT sheets, MWNT yarns, or standard 2-dimensional culture conditions as a control. We find comparable cell viability between all three culture conditions but enhanced production of the hepatocyte-specific marker albumin for cells cultured on MWNTs. The basal activity of two clinically relevant cytochrome P450 enzymes, CYP1A2 and CYP3A4, are similar on all substrates, but we find enhanced induction of CYP1A2 for cells on the MWNT sheets. Our data thus supports the use of these substrates for applications including tissue engineering and enhancing liver-specific functions, as well as in in vitro model systems with enhanced predictive capability in drug discovery and development.

  18. A defined, controlled culture system for primary bovine chromaffin progenitors reveals novel biomarkers and modulators.

    PubMed

    Masjkur, Jimmy; Levenfus, Ian; Lange, Sven; Arps-Forker, Carina; Poser, Steve; Qin, Nan; Vukicevic, Vladimir; Chavakis, Triantafyllos; Eisenhofer, Graeme; Bornstein, Stefan R; Ehrhart-Bornstein, Monika; Androutsellis-Theotokis, Andreas

    2014-07-01

    We present a method to efficiently culture primary chromaffin progenitors from the adult bovine adrenal medulla in a defined, serum-free monolayer system. Tissue is dissociated and plated for expansion under support by the mitogen basic fibroblast growth factor (bFGF). The cultures, although not homogenous, contain a subpopulation of cells expressing the neural stem cell marker Hes3 that also propagate. In addition, Hes3 is also expressed in the adult adrenal medulla from where the tissue is taken. Differentiation is induced by bFGF withdrawal and switching to Neurobasal medium containing B27. Following differentiation, Hes3 expression is lost, and cells acquire morphologies and biomarker expression patterns of chromaffin cells and dopaminergic neurons. We tested the effect of different treatments that we previously showed regulate Hes3 expression and cell number in cultures of fetal and adult rodent neural stem cells. Treatment of the cultures with a combination of Delta4, Angiopoietin2, and a Janus kinase inhibitor increases cell number during the expansion phase without significantly affecting catecholamine content levels. Treatment with cholera toxin does not significantly affect cell number but reduces the ratio of epinephrine to norepinephrine content and increases the dopamine content relative to total catecholamines. These data suggest that this defined culture system can be used for target identification in drug discovery programs and that the transcription factor Hes3 may serve as a new biomarker of putative adrenomedullary chromaffin progenitor cells.

  19. Changes in adipose tissue stromal-vascular cells in primary culture due to porcine sera

    SciTech Connect

    Jewell, D.E.; Hausman, G.J.

    1986-03-01

    This study was conducted to determine the response of rat stromal-vascular cells to pig sea. Sera were collected from unselected contemporary (lean) and high backfat thickness selected (obese) pigs. Sera from obese pigs were collected either by exsanguination or cannulation. sera from lean pigs during the growing phase (45 kg) and the fattening phase (100-110 kg) were collected. Stromal-vascular cells derived rom rat inguinal tissue were cultured on either 25 cm/sup 2/ flasks, collagen-coated coverslips or petri dishes. Cell proliferation was measured by (/sup 3/H)-thymidine incorporation during the fourth day of culture. Coverslip cultures were used for histochemical analysis. Petri dish cultures were used for analysis of Sn-glycerol-3-phosphate dehydrogenase (GPDH) activity. All cells were plated for 24 hours in media containing 10 fetal bovine sera. Test media contained 2.5, 5.0, 10.0% sera. Sera from obese pigs increased GPDH activity and fat cell production when compared to the lean controls. The increased concentration of sera increased esterase activity and lipid as measured with oil red O. The sera from obese pigs collected at slaughter stimulated more fat cell production than obese sera collected by cannulation. These studies show there are adipogenic factors in obese pigs sera which promote fat cell development in primary cell culture.

  20. Primary possum macrophage cultures support the growth of a nidovirus associated with wobbly possum disease.

    PubMed

    Giles, Julia C; Perrott, Matthew R; Dunowska, Magdalena

    2015-09-15

    The objective of the study was to establish a system for isolation of a recently described, thus far uncultured, marsupial nidovirus associated with a neurological disease of possums, termed wobbly possum disease (WPD). Primary cultures of possum macrophages were established from livers of adult Australian brushtail possums (Trichosurus vulpecula). High viral copy numbers (up to 6.9×10(8)/mL of cell lysate) were detected in infected cell culture lysates from up to the 5th passage of the virus, indicating that the putative WPD virus (WPDV) was replicating in cultured cells. A purified virus stock with a density of 1.09 g/mL was prepared using iodixanol density gradient ultracentrifugation. Virus-like particles approximately 60 nm in diameter were observed using electron microscopy in negatively stained preparations of the purified virus. The one-step growth curve of WPDV in macrophage cultures showed the highest increase in intracellular viral RNA between 6 and 12h post-infection. Maximum levels of cell-associated viral RNA were detected at 24h post-infection, followed by a decline. Levels of extracellular RNA increased starting at 9h post-infection, with maximum levels detected at 48 h post-infection. The establishment of the in vitro system to culture WPDV will facilitate further characterisation of this novel nidovirus.

  1. Primary Culture of Mycobacterium ulcerans from Human Tissue Specimens after Storage in Semisolid Transport Medium▿

    PubMed Central

    Eddyani, Miriam; Debacker, Martine; Martin, Anandi; Aguiar, Julia; Johnson, Christian R.; Uwizeye, Cécile; Fissette, Krista; Portaels, Françoise

    2008-01-01

    Tissue specimens collected from patients with clinically suspected Buruli ulcer treated in two Buruli ulcer treatment centers in Benin between 1998 and 2004 were placed in semisolid transport medium and transported at ambient temperature for microbiological analysis at the Institute of Tropical Medicine in Antwerp, Belgium. The impact of the delay before microbiological analysis on primary culture of Mycobacterium ulcerans was investigated. The length of storage in semisolid transport medium varied from 6 days to 26 weeks. Of the 1,273 tissue fragments positive for M. ulcerans DNA by an IS2404-specific PCR, 576 (45.2%) yielded positive culture results. The sensitivity of direct smear examination was 64.6% (822/1,273 tissue fragments). The median time required to obtain a positive culture result was 11 weeks. Positive cultures were obtained even from samples kept for more than 2 months at ambient temperatures. Moreover, there was no reduction in the viability of M. ulcerans, as detected by culture, when specimens remained in semisolid transport medium for long periods of time (up to 26 weeks). We can conclude that the method with semisolid transport medium is very robust for clinical specimens from patients with Buruli ulcer that, due to circumstances, cannot be analyzed in a timely manner. This transport medium is thus very useful for the confirmation of a diagnosis of Buruli ulcer with specimens collected in the field. PMID:17989199

  2. The implementation of a social constructivist approach in primary science education in Confucian heritage culture: the case of Vietnam

    NASA Astrophysics Data System (ADS)

    Hằng, Ngô Vũ Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

    2015-09-01

    Social constructivism has been increasingly studied and implemented in science school education. Nevertheless, there is a lack of holistic studies on the implementation of social constructivist approach in primary science education in Confucian heritage culture. This study aims to determine to what extent a social constructivist approach is implemented in primary science education in Confucian heritage culture and to give explanations for the implementation from a cultural perspective. Findings reveal that in Confucian heritage culture a social constructivist approach has so far not implemented well in primary science education. The implementation has been considerably influenced by Confucian heritage culture, which has characteristics divergent from and aligning with those of social constructivism. This study indicates a need for design-based research on social constructivism-based science curriculum for Confucian heritage culture.

  3. The effect of anabolic-androgenic steroids on primary myocardial cell cultures.

    PubMed

    Melchert, R B; Herron, T J; Welder, A A

    1992-02-01

    Although recent case reports suggest that anabolic-androgenic steroids may be directly injurious to the cardiovascular system, the direct myocardial cellular consequences of abuse of these drugs are not known. Therefore, the purpose of this study was to describe the concentration- and time-dependent effects of testosterone cypionate (TC), stanozolol (S), and fluoxymesterone (F) on primary myocardial cell cultures. Evaluation of drug effects were made in 4-d-old primary myocardial cell cultures obtained from 3- to 5-d-old Sprague-Dawley rats. The cultures were exposed to 1 x 10(-4) M, 1 x 10(-6) M, and 1 x 10(-8) M concentrations of TC, S, and F each for 1, 4, and 24 h. Cellular injury was evaluated by alterations in beating activity, induction of morphological alterations, lactate dehydrogenase (LDH) release, neutral red retention, and tetrazolium (MTT) formazan production. Significant alterations in beating activity were observed in the 1 x 10(-4) M TC group in which no beating activity was seen at 1, 4, and 24 h. Morphological integrity was disrupted for the 1 x 10(-4) M TC group at 24 h where destruction of the monolayer was observed. Unlike the cultures treated with the three concentrations of both S and F, significant LDH release was seen at 4 and 24 h with those cultures exposed to 1 x 10(-4) M TC. In the evaluation of neutral red retention, 1 x 10(-4) M TC at 24 h showed a significant decrease in ability to retain the dye.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Atherosclerosis- and age-related multinucleated variant endothelial cells in primary culture from human aorta.

    PubMed Central

    Tokunaga, O.; Fan, J. L.; Watanabe, T.

    1989-01-01

    Endothelial cells were cultured from human aortas and inferior venae cavae of autopsied subjects ranging in age from infancy to 85 years. Endothelial cells in 32 of more than 100 attempted cultures were pure enough for evaluation. Emerged endothelial cells in primary culture were classified into two types: typical endothelium and variant endothelium. Typical endothelial cells were small, round to polygonal shaped, and were arranged uniformly. Their diameter ranged from 50 to 70 microns. Variant endothelial cells were larger, ranging from 100 to 200 microns in diameter, and giant endothelial cells measuring more than 250 microns in diameter were scattered among them. Variant endothelial cells were usually multinucleated and possessed endothelium-specific markers of vWF and Weibel-Palade bodies. No incorporation of [3H]thymidine was found in the nuclei of cultured variant endothelial cells. Although most cultured endothelial cells were of the typical type, variant endothelial cells were interspersed throughout the culture. The ratio of variant endothelial cells to typical cells correlated well with the severity of atherosclerosis, but less so with aging. The number of variant endothelial cells in cultures from inferior venae cavae was slight and constant throughout all age groups. The presence of multinucleated endothelial cells in in vivo aortas was confirmed by both scanning and transmission electron microscopy. They sometimes existed in colonies in the aortas from elderly subjects with intimal-thickened or advanced atherosclerotic lesions. These results indicate that variant endothelial cells were present in vivo and their ratio in primary culture reflected the in vivo population. It is likely that these cells were formed by adhesion of adjacent typical endothelial cells and that this process was affected more by atherosclerosis than by aging. Although it is not clear if the multinucleated variant cells were formed before the formation of atherosclerotic plaque or

  5. Between orientalism and normalization: cross-cultural lessons from Japan for a critical history of psychology.

    PubMed

    Burman, Erica

    2007-05-01

    Cross-cultural research performs a vital role within the confirmation of psychological "truths." Its differentiations work simultaneously to establish their general applicability and the superiority of Anglo-U.S. ways of living and relating. Taking three examples of how "Japan" figures within English language psychological accounts (i.e., group/individual, shame/guilt societies, and attachment styles), I indicate how the apparent stability of these truths suppressed the violent history of their generation. Moreover, I suggest how resisting the assimilation of cultural specificity into a discourse of mere variation can challenge the hegemony of Anglo-U.S. psychology and reframe the vexed question of specificity versus universality.

  6. Tissue Factor Activity in Lymphocyte Cultures from Normal Individuals and Patients with Hemophilia A

    PubMed Central

    Rickles, Frederick R.; Hardin, John A.; Pitlick, Frances A.; Hoyer, Leon W.; Conrad, Marcel E.

    1973-01-01

    The procoagulant material of lymphocytes has been characterized as tissue factor. Lymphocytes stimulated with phytohemagglutinin or the purified protein derivative of the tubercle bacillus developed procoagulant activity with incubation in tissue culture. While this material corrected the prolonged clotting time of factor VIII (AHF) deficient plasma, we have shown, utilizing a sensitive radioimmunoassay, that no AHF antigen was present in the cell cultures. Further, we have demonstrated this material to be tissue factor by coagulation techniques and immunological cross-reactivity. The published data regarding factor VIII synthesis is reviewed in light of these observations and comments are made regarding the role of the lymphocyte procoagulant. PMID:4634046

  7. [Use of hepatocytes in primary cultures to predict potential hepatocarcinogenicity of compounds

    PubMed

    Bieri, F.; Muakkassah-Kelly, S.; Bentley, P.

    1990-01-01

    Short-term tests for genotoxic activity will detect a variety of potential carcinogens. However, experience over the last few years has shown that many chemical carcinogens are not detected in these tests. A large proportion of these non-mutagenic (non-genotoxic) carcinogens induce tumors in the rodent liver after life-long feeding. One class of such carcinogens are the hypolipidemic drugs many of which induce a characteristic hepatomegaly upon subchronic treatment. Typically this liver enlargement is accompanied by increased mitotic activity and a proliferation of the hepatic peroxisome compartment. Results also suggest a correlation between the degree and the nature of the hepatomegalic response to a compound, and its hepatocarcinogenic potency, but it remains unclear whether the growth stimulation and/or the peroxisome proliferation is correlated with the carcinogenicity. Both, the induction of peroxisomal enzyme and of replicative DMA synthesis may be measured in primary cultures of adult rat hepatocytes following in vitro exposition. Moreover, the ability of the compounds tested to induce in vitro replicative DNA synthesis in primary cultures of adult rat hepatocytes correlated well with the potency of the hepatomegalic response induced in vivo in treated rodents. Consequently, studies using such cultures might be useful to characterize and possibly predict in vitro the hepatocarcinogenic potency of some new compounds. The early recognition of the possible carcinogenic property of a new substance might accordingly contribute to the reduction of a number of life-long studies.

  8. Basal cells are the progenitors of primary tracheal epithelial cell cultures

    SciTech Connect

    Ford, J.R.; Terzaghi-Howe, M. Oak Ridge National Lab., TN )

    1992-01-01

    The goal of this study was to identify the cells from the rat tracheal epithelium which attach and proliferate in primary culture. When cells isolated from tracheas by enzymatic digestion were held in suspension at 37C for several hours most of the differentiated cells dies. The kinetics of this selective cell death were not dependent on the constituents of the holding medium. With time in suspension, the colony forming efficiency of the surviving cells increased two- to threefold. Comparison of the growth curves of cells held or plated directly showed no difference in the number of cells in the proliferating populations. Using two lectins, it was possible to monitor the loss of specific populations in suspension. BS1-B4 is a marker for basal cells and UEA-1 is a secretory cell marker. Only those cells that were BS1-B4 positive survived in suspension. Further, the colonies that formed in primary culture were positive for this marker. Single cell suspensions of cells were sorted by flow cytometry and a fivefold increase in the colony forming efficiency of BS1-B4 positive cells compared to that of the negative cells was observed. These findings suggest that the cells that survived in suspension and proliferated in culture originated from the basal cells of the trachea.

  9. Establishment of primary cultures for mouse ameloblasts as a model of their lifetime

    SciTech Connect

    Suzawa, Tetsuo . E-mail: suzawa@dent.showa-u.ac.jp; Itoh, Nao; Takahashi, Naoyuki; Katagiri, Takenobu; Morimura, Naoko; Kobayashi, Yasuna; Yamamoto, Toshinori; Kamijo, Ryutaro

    2006-07-07

    To understand how the properties of ameloblasts are spatiotemporally regulated during amelogenesis, two primary cultures of ameloblasts in different stages of differentiation were established from mouse enamel epithelium. Mouse primary ameloblasts (MPAs) prepared from immature enamel epithelium (MPA-I) could proliferate, whereas those from mature enamel epithelium (MPA-M) could not. MPA-M but not MPA-I caused apoptosis during culture. The mRNA expression of amelogenin, a marker of immature ameloblasts, was down-regulated, and that of enamel matrix serine proteiase-1, a marker of mature ameloblasts, was induced in MPA-I during culture. Using green fluorescence protein as a reporter, a visualized reporter system was established to analyze the promoter activity of the amelogenin gene. The region between -1102 bp and -261 bp was required for the reporter expression in MPA-I. These results suggest that MPAs are valuable in vitro models for investigation of ameloblast biology, and that the visualized system is useful for promoter analysis in MPAs.

  10. Osmotic water permeability diversification in primary trophoblast cultures from aquaporin 1-deficient pregnant mice.

    PubMed

    Sha, Xiao-Yan; Liu, Hui-Shu; Ma, Tong-Hui

    2015-09-01

    Aquaporins (AQP) are water channel proteins, and some play an important role in maternal-fetal fluid exchange. The present study aimed to measure the osmotic water permeability in primary cultures of trophoblast cells from AQP1-deficient (AQP1(-/-) ) pregnant mice and to define the quantitative role of AQP1 in water transport across the trophoblast plasma membrane. Trophoblast cells were obtained from placental tissue cell culture of AQP1(-/-) pregnant mice and were characterized by cytokeratin 7 immunostaining. The expression of the AQP1 gene in trophoblast cells of wild-type (AQP1(+/+) ) mice was confirmed by immunofluorescence. The osmotic water permeability of trophoblast plasma membranes was measured by a calcein fluorescence quenching method in response to osmotic gradients. A primary cell culture system for trophoblasts was successfully established. Immunofluorescence showed the expression of AQP1 in the trophoblast cell membrane of AQP1(+/+) mice. The osmotic water permeability of AQP1(-/-) trophoblast cells was significantly lower than that in AQP1(+/+) trophoblast cells, in response to both hypotonic and hypertonic challenges. The results suggest an important role of AQP1-mediated plasma membrane water permeability in maternal-fetal fluid balance and also provide a potential direction for the identification of therapeutic targets for the treatment of abnormalities in amniotic fluid volume. © 2015 Japan Society of Obstetrics and Gynecology.

  11. Sugammadex, a neuromuscular blockade reversal agent, causes neuronal apoptosis in primary cultures.

    PubMed

    Palanca, José M; Aguirre-Rueda, Diana; Granell, Manuel V; Aldasoro, Martin; Garcia, Alma; Iradi, Antonio; Obrador, Elena; Mauricio, Maria Dolores; Vila, Jose; Gil-Bisquert, Anna; Valles, Soraya L

    2013-01-01

    Sugammadex, a γ-cyclodextrin that encapsulates selectively steroidal neuromuscular blocking agents, such as rocuronium or vecuronium, has changed the face of clinical neuromuscular pharmacology. Sugammadex allows a rapid reversal of muscle paralysis. Sugammadex appears to be safe and well tolerated. Its blood-brain barrier penetration is poor (< 3% in rats), and thus no relevant central nervous toxicity is expected. However the blood brain barrier permeability can be altered under different conditions (i.e. neurodegenerative diseases, trauma, ischemia, infections, or immature nervous system). Using MTT, confocal microscopy, caspase-3 activity, cholesterol quantification and Western-blot we determine toxicity of Sugammadex in neurons in primary culture. Here we show that clinically relevant sugammadex concentrations cause apoptotic/necrosis neuron death in primary cultures. Studies on the underlying mechanism revealed that sugammadex-induced activation of mitochondria-dependent apoptosis associates with depletion of neuronal cholesterol levels. Furthermore SUG increase CytC, AIF, Smac/Diablo and CASP-3 protein expression in cells in culture. Potential association of SUG-induced alteration in cholesterol homeostasis with oxidative stress and apoptosis activation occurs. Furthermore, resistance/sensitivity to oxidative stress differs between neuronal cell types.

  12. Sugammadex, a Neuromuscular Blockade Reversal Agent, Causes Neuronal Apoptosis in Primary Cultures

    PubMed Central

    Palanca, José M.; Aguirre-Rueda, Diana; Granell, Manuel V.; Aldasoro, Martin; Garcia, Alma; Iradi, Antonio; Obrador, Elena; Mauricio, Maria Dolores; Vila, Jose; Gil-Bisquert, Anna; Valles, Soraya L.

    2013-01-01

    Sugammadex, a γ-cyclodextrin that encapsulates selectively steroidal neuromuscular blocking agents, such as rocuronium or vecuronium, has changed the face of clinical neuromuscular pharmacology. Sugammadex allows a rapid reversal of muscle paralysis. Sugammadex appears to be safe and well tolerated. Its blood-brain barrier penetration is poor (< 3% in rats), and thus no relevant central nervous toxicity is expected. However the blood brain barrier permeability can be altered under different conditions (i.e. neurodegenerative diseases, trauma, ischemia, infections, or immature nervous system). Using MTT, confocal microscopy, caspase-3 activity, cholesterol quantification and Western-blot we determine toxicity of Sugammadex in neurons in primary culture. Here we show that clinically relevant sugammadex concentrations cause apoptotic/necrosis neuron death in primary cultures. Studies on the underlying mechanism revealed that sugammadex-induced activation of mitochondria-dependent apoptosis associates with depletion of neuronal cholesterol levels. Furthermore SUG increase CytC, AIF, Smac/Diablo and CASP-3 protein expression in cells in culture. Potential association of SUG-induced alteration in cholesterol homeostasis with oxidative stress and apoptosis activation occurs. Furthermore, resistance/sensitivity to oxidative stress differs between neuronal cell types. PMID:23983586

  13. Dual signal transduction pathways activated by TSH receptors in rat primary tanycyte cultures.

    PubMed

    Bolborea, Matei; Helfer, Gisela; Ebling, Francis J P; Barrett, Perry

    2015-06-01

    Tanycytes play multiple roles in hypothalamic functions, including sensing peripheral nutrients and metabolic hormones, regulating neurosecretion and mediating seasonal cycles of reproduction and metabolic physiology. This last function reflects the expression of TSH receptors in tanycytes, which detect photoperiod-regulated changes in TSH secretion from the neighbouring pars tuberalis. The present overall aim was to determine the signal transduction pathway by which TSH signals in tanycytes. Expression of the TSH receptor in tanycytes of 10-day-old Sprague Dawley rats was observed by in situ hybridisation. Primary ependymal cell cultures prepared from 10-day-old rats were found by immunohistochemistry to express vimentin but not GFAP and by PCR to express mRNA for Dio2, Gpr50, Darpp-32 and Tsh receptors that are characteristic of tanycytes. Treatment of primary tanycyte/ependymal cultures with TSH (100  IU/l) increased cAMP as assessed by ELISA and induced a cAMP-independent increase in the phosphorylation of ERK1/2 as assessed by western blot analysis. Furthermore, TSH (100  IU/l) stimulated a 2.17-fold increase in Dio2 mRNA expression. We conclude that TSH signal transduction in cultured tanycytes signals via Gαs to increase cAMP and via an alternative G protein to increase phosphorylation of ERK1/2.

  14. Primary culture of gustatory receptor neurons from the blowfly, Phormia regina.

    PubMed

    Murata, Yoshihiro; Ozaki, Mamiko; Nakamura, Tadashi

    2006-07-01

    Flies provide a powerful model system for exploring signaling systems in gustatory receptor neurons (GRNs). To elucidate the cellular and molecular bases of these signaling systems, we sought to develop techniques to dissociate GRNs. We developed a primary culture of GRNs isolated from the labella of the blowfly, Phormia regina, 4-5 days after pupation. Dissected labella were treated with papain in a low Ca2+ saline solution and shaken in Leibovitz's L-15 medium supplemented with 20-hydroxyecdysone, L-ascorbic acid, and trehalose with a test tube mixer. Released cells were plated and kept at 29 degrees C in a medium containing fetal bovine serum. After a minimum of 2 days in culture, we observed survival or growth of bipolar cells with the characteristic morphology of GRNs. We also examined taste responsiveness by monitoring intracellular Ca2+ with a Ca2+-sensitive fluorescent dye, fluo-3. For some bipolar cells, application of sucrose, NaCl, or LiCl for 5-20 s transiently increased the intracellular Ca2+ levels in cell bodies for 20-30 s. The primary cell culture described here is useful for functional analysis of GRNs.

  15. Tumor necrosis factor (cachetin) decreases adipose cell differentiation in primary cell culture

    SciTech Connect

    Martin, R.J.; Jones, D.D.; Jewell, D.E.; Hausman, G.J.

    1986-03-05

    Cachetin has been shown to effect gene product expression in the established adipose cell line 3T3-L1. Expression of messenger RNA for lipoprotein lipase is suppressed in cultured adipocytes. The purpose of this study was to determine the effect of Cachetin on adipose cell differentiation in primary cell culture. Stromalvascular cells obtained from the inguinal fat pad of 4-5 week old Sprague-Dawley rats were grown in culture for two weeks. During the proliferative growth phase all cells were grown on the same medium and labelled with /sup 3/H-thymidine. Cachetin treatment (10/sup -6/ to 10/sup -10/ M) was initiated on day 5, the initial phase of preadipocyte differentiation. Adipocytes and stromal cells were separated using density gradient, and /sup 3/H-thymidine was determined for both cell types. Thymidine incorporation into adipose cells was decreased maximally (approx. 50%) at 10/sup -10/ M. Stromalvascular cells were not influenced at any of the doses tested. Adipose cell lipid content as indicated by oil red-O staining was decreased by Cachetin. Esterase staining by adipose cells treated with Cachetin was increased indicating an increase in intracellular lipase. These studies show that Cachetin has specific effects on primary adipose cell differentiation.

  16. Differentiation of primary human submandibular gland cells cultured on basement membrane extract.

    PubMed

    Szlávik, Vanda; Szabó, Bálint; Vicsek, Tamás; Barabás, József; Bogdán, Sándor; Gresz, Veronika; Varga, Gábor; O'Connell, Brian; Vág, János

    2008-11-01

    There is no effective treatment for the loss of functional salivary tissue after irradiation for head and neck cancer or the autoimmune disease Sjögren's syndrome. One possible approach is the regeneration of salivary glands from stem cells. The present study aimed to investigate whether small pieces of human submandiblar gland tissue contain elements necessary for the reconstruction of salivary rudiments in vitro via acinar and ductal cell differentiation. Primary submandibular gland (primary total human salivary gland; PTHSG) cells were isolated from human tissue and cultured in vitro using a new method in which single cells form an expanding epithelial monolayer on plastic substrates. Differentiation, morphology, number, and organization of these cells were then followed on basement membrane extract (BME) using RNA quantitation (amylase, claudin-1 (CLN1), CLN3, kallikrein, vimentin), immunohistochemistry (amylase and occludin), viability assay, and videomicroscopy. On the surface of BME, PTHSG cells formed acinotubular structures within 24 h, did not proliferate, and stained for amylase. In cultures derived from half of the donors, the acinar markers amylase and CLN3 were upregulated. The PTHSG culture model suggests that human salivary gland may be capable of regeneration via reorganization and differentiation and that basement membrane components play a crucial role in the morphological and functional differentiation of salivary cells.

  17. Cultural Reciprocity in Sociocultural Perspective: Adapting the Normalization Principle for Family Collaboration.

    ERIC Educational Resources Information Center

    Harry, Beth; Rueda, Robert; Kalyanpur, Maya

    1999-01-01

    Findings from a collaborative action research project involving seven culturally diverse families with children with disabilities are used to illustrate how professionals can provide assistance in a family's zone of proximal development, rather than targeting goals that are normative for the mainstream, but not for the family. (Author/CR)

  18. Cultural Reciprocity in Sociocultural Perspective: Adapting the Normalization Principle for Family Collaboration.

    ERIC Educational Resources Information Center

    Harry, Beth; Rueda, Robert; Kalyanpur, Maya

    1999-01-01

    Findings from a collaborative action research project involving seven culturally diverse families with children with disabilities are used to illustrate how professionals can provide assistance in a family's zone of proximal development, rather than targeting goals that are normative for the mainstream, but not for the family. (Author/CR)

  19. Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice.

    PubMed

    Hindi, Lubna; McMillan, Joseph D; Afroze, Dil; Hindi, Sajedah M; Kumar, Ashok

    2017-05-05

    Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many steps of myogenesis can be recapitulated through in vitro differentiation of myoblasts into myotubes. Most laboratories use immortalized myogenic cells lines that also differentiate into myotubes. Although these cell lines have been found quite useful to delineating the regulatory mechanisms of myogenesis, they often show a great degree of variability depending on the origin of the cells and culture conditions. Primary myoblasts have been suggested as the most physiologically relevant model for studying myogenesis in vitro. However, due to their low abundance in adult skeletal muscle, isolation of primary myoblasts is technically challenging. In this article, we describe an improved protocol for the isolation of primary myoblasts from adult skeletal muscle of mice. We also describe methods for their culturing and differentiation into myotubes.

  20. Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice

    PubMed Central

    Hindi, Lubna; McMillan, Joseph D.; Afroze, Dil; Hindi, Sajedah M.; Kumar, Ashok

    2017-01-01

    Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many steps of myogenesis can be recapitulated through in vitro differentiation of myoblasts into myotubes. Most laboratories use immortalized myogenic cells lines that also differentiate into myotubes. Although these cell lines have been found quite useful to delineating the regulatory mechanisms of myogenesis, they often show a great degree of variability depending on the origin of the cells and culture conditions. Primary myoblasts have been suggested as the most physiologically relevant model for studying myogenesis in vitro. However, due to their low abundance in adult skeletal muscle, isolation of primary myoblasts is technically challenging. In this article, we describe an improved protocol for the isolation of primary myoblasts from adult skeletal muscle of mice. We also describe methods for their culturing and differentiation into myotubes. PMID:28730161

  1. Cerium oxide nanoparticles prevent apoptosis in primary cortical culture by stabilizing mitochondrial membrane potential.

    PubMed

    Arya, A; Sethy, N K; Das, M; Singh, S K; Das, A; Ujjain, S K; Sharma, R K; Sharma, M; Bhargava, K

    2014-07-01

    Cerium oxide nanoparticles (CNPs) of spherical shape have unique antioxidant capacity primarily due to alternating + 3 and + 4 oxidation states and crystal defects. Several studies revealed the protective efficacies of CNPs in cells and tissues against the oxidative damage. However, its effect on mitochondrial functioning, downstream effectors of radical burst and apoptosis remains unknown. In this study, we investigated whether CNPs treatment could protect the primary cortical cells from loss of mitochondrial membrane potential (Δψm) and Δψm-dependent cell death. CNPs with spherical morphology and size range 7-10 nm were synthesized and utilized at a concentration of 25 nM on primary neuronal culture challenged with 50 μM of hydrogen peroxide (H2O2). We showed that optimal dose of CNPs minimized ROS content of the cells and also curbed related surge in cellular calcium flux. Importantly, CNPs treatment prevented apoptotic loss of cell viability. Reduction in the apoptosis could be successfully attributed to the maintenance of Δψm and restoration of major redox equivalents NADH/NAD(+) ratio and cellular ATP. These findings, therefore, suggest possible route of CNPs protective efficacies in primary cortical culture.

  2. Cinematographic observations of growth cycles of Chlamydia trachomatis in primary cultures of human amniotic cells.

    PubMed Central

    Neeper, I D; Patton, D L; Kuo, C C

    1990-01-01

    Time-lapse cinematography was used to study the growth cycle of Chlamydia trachomatis in primary cell cultures of human amnion. Twelve preterm and twelve term placentas were obtained within 8 h of delivery, and epithelial cells were dissociated from the amniotic membranes by trypsinization and grown in Rose chambers. The epithelial nature of the cultured cells was documented by morphology and by immunofluorescence staining for cytoskeletal proteins, which matched the staining of intact amnion. With regular feedings, uninfected cultures remained healthy for up to 30 days. Confluent cultures (7 to 10 days) were infected with a genital strain (E/UW-5/CX) of C. trachomatis at 10(5) infectious units per chamber. Infections were done in culture medium without cycloheximide, which is often used to induce susceptibility of the cells. Between 66 and 90% of the cells were infected. Intracytoplasmic inclusions were visible by 18 h post infection (p.i.) and grew larger as the organisms inside multiplied. By 72 h p.i., the inclusions occupied the entire cytoplasm of the host cells. Further growth of the inclusions overdistended and ruptured the host cells on days 3 to 7. Cells not infected by the original inoculum became infected on day 5 or 6 p.i. by the chlamydial particles released from the ruptured cells. No amniotic cell was ever observed to survive the infection. The data presented support the hypothesis that amniotic epithelium is susceptible to infection and damage by C. trachomatis. This culture system provided detailed and dynamic observations of chlamydial infection under conditions more nearly physiologic than previously reported. Images PMID:2365450

  3. Plaque Morphology of Teschen Disease Viruses and Certain Pig Enteroviruses in Primary pig Kidney Monolayer Cultures

    PubMed Central

    Dardiri, A. H.

    1968-01-01

    Plaque patterns and diameters of four virulent strains and one tissue culture mutant of Teschen disease virus were compared with six pig enteroviruses isolated in the United States. They are described as they were produced in primary pig kidney monolayer cultures. Reproducible plaques, with similar characteristics and class-types of each of the viruses tested were obtained with the application of a 45-minute virus adsorption time. Their morphologic characteristics and the proportion in which the plaque types appeared may assist in the differentiation of these virus strains. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 7.Fig. 8.Fig. 9.Fig. 10.Fig. 11.Fig. 12. PMID:4299823

  4. Using homogeneous primary neuron cultures to study fundamental aspects of HSV-1 latency and reactivation.

    PubMed

    Kim, Ju Youn; Shiflett, Lora A; Linderman, Jessica A; Mohr, Ian; Wilson, Angus C

    2014-01-01

    We describe a primary neuronal culture system suitable for molecular characterization of herpes simplex virus type 1 (HSV-1) infection, latency, and reactivation. While several alternative models are available, including infections of live animal and explanted ganglia, these are complicated by the presence of multiple cell types, including immune cells, and difficulties in manipulating the neuronal environment. The highly pure neuron culture system described here can be readily manipulated and is ideal for molecular studies that focus exclusively on the relationship between the virus and host neuron, the fundamental unit of latency. As such it allows for detailed investigations of both viral and neuronal factors involved in the establishment and maintenance of HSV-1 latency and in viral reactivation induced by defined stimuli.

  5. Genetic basis of triticale breeding (x triticale). IV. Embryo culture for synthesizing primary hexaploid triticales

    SciTech Connect

    Gordei, I.A.; Khodortsova, L.F.

    1986-07-01

    Results are reported on enhancing the efficiency of embryo culture for synthesizing primary hexaploid triticales (AABBRR, 2n = 42). The antioxidant tomatoside has a positive effect on the reduction of progamous incompatibility of wheat with rye and increases the output of wheat-rye amphihaploids. It has been established that irradiation of embryos, cultured on nutrient medium, with helium-neon laser, increases significantly (P < 0.01) the regeneration frequency of the wheat-rye hybrid embryos. The highest frequency (40%) of amphidiploids was obtained by treating the plants with 0.15% colchicine through roots during the tillering phase. Hexaploid triticales from 11 cross combinations between tetraploid wheats (AABB, 2n = 28) and diploid rye (RR, 2n = 14) formed the initial material for breeding.

  6. Betulin elicits anti-cancer effects in tumour primary cultures and cell lines in vitro.

    PubMed

    Rzeski, Wojciech; Stepulak, Andrzej; Szymański, Marek; Juszczak, Małgorzata; Grabarska, Aneta; Sifringer, Marco; Kaczor, Józef; Kandefer-Szerszeń, Martyna

    2009-12-01

    Betulin is a pentacyclic triterpene found in many plant species, among others, in white birch bark. The aim of the study was in vitro characterization of the anticancer activity of betulin in a range of human tumour cell lines (neuroblastoma, rhabdomyosarcoma-medulloblastoma, glioma, thyroid, breast, lung and colon carcinoma, leukaemia and multiple myeloma), and in primary tumour cultures isolated from patients (ovarian carcinoma, cervical carcinoma and glioblastoma multiforme). In this study, we demonstrated a remarkable anti-proliferative effect of betulin in all tested tumour cell cultures. Neuroblastoma (SK-N-AS) and colon carcinoma (HT-29) were the most sensitive to the anti-proliferative effect of betulin. Furthermore, betulin altered tumour cells morphology, decreased their motility and induced apoptotic cell death. These findings demonstrate the anti-cancer potential of betulin and suggest that they may be applied as an adjunctive measure in cancer treatment.

  7. Utilization of supplemental methionine sources by primary cultures of chick hepatocytes

    SciTech Connect

    Dibner, J.J.

    1983-10-01

    Utilization of 2-hydroxy-4-(methylthio) butanoic acid (HMB) as a substrate for protein synthesis was studied by using primary cultures of chick liver cells. Cultures were prepared by enzymatic dissociation of livers from week old Hubbard broiler chicks and were maintained for 4 days under nonproliferative conditions. Hepatocyte differentiation was verified by using dexamethasone induction of tyrosine aminotransferase activity. Conversion of (14C)HMB to L-methionine was shown by chromatographic analysis of hepatocyte protein hydrolysate and incorporation into protein was proven by cycloheximide inhibition of synthesis. When incorporation of HMB was compared to that of DL-methionine (DLM) equimolar quantities of the two sources were found in liver cell protein. These results support, at a cellular level, the conclusion that HMB and DLM are biochemically equivalent sources of methionine for protein synthesis.

  8. Primary care units in Emilia-Romagna, Italy: an assessment of organizational culture.

    PubMed

    Pracilio, Valerie P; Keith, Scott W; McAna, John; Rossi, Giuseppina; Brianti, Ettore; Fabi, Massimo; Maio, Vittorio

    2014-01-01

    This study investigates the organizational culture and associated characteristics of the newly established primary care units (PCUs)-collaborative teams of general practitioners (GPs) who provide patients with integrated health care services-in the Emilia-Romagna Region (RER), Italy. A survey instrument covering 6 cultural dimensions was administered to all 301 GPs in 21 PCUs in the Local Health Authority (LHA) of Parma, RER; the response rate was 79.1%. Management style, organizational trust, and collegiality proved to be more important aspects of PCU organizational culture than information sharing, quality, and cohesiveness. Cultural dimension scores were positively associated with certain characteristics of the PCUs including larger PCU size and greater proportion of older GPs. The presence of female GPs in the PCUs had a negative impact on collegiality, organizational trust, and quality. Feedback collected through this assessment will be useful to the RER and LHAs for evaluating and guiding improvements in the PCUs. © 2013 by the American College of Medical Quality.

  9. Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

    PubMed Central

    Pilgrim, Matthew G.; Lengyel, Imre; Lanzirotti, Antonio; Newville, Matt; Fearn, Sarah; Emri, Eszter; Knowles, Jonathan C.; Messinger, Jeffrey D.; Read, Russell W.; Guidry, Clyde; Curcio, Christine A.

    2017-01-01

    Purpose Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Methods Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Results Apparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. Conclusions The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss. PMID:28146236

  10. Effect of acrylamide-induced neurotoxicity in a primary astrocytes/microglial co-culture model.

    PubMed

    Zhao, Mengyao; Wang, Fu Sheng Lewis; Hu, Xiao Song; Chen, Fang; Chan, Hing Man

    2017-03-01

    Acrylamide (AA), is a common food contaminant generated by heat processing. Astrocytes and microglia are the two major glial cell types in the brain that play pivotal but different roles in maintaining optimal brain function. The objective of this study is to investigate the neurotoxicity of AA, using a primary astrocytes/microglia co-culture model. Co-cultural cells obtained from Balb/c mice were cultured and treated with 0-1.0mM AA for 24-96h. Cell viability, reactive oxygen species (ROS) generation, oxidative end produces formation and glutathione (GSH) levels were measured. The expression of nuclear-E2-related factor 2(Nrf2), and nuclear factor kappa-beta (NF-κB) and selected down-stream genes were measured. Results showed that AA treatment led toa dose-dependent toxicity. Oxidative stress was induced as indicated by an increase of ROS, a decrease of GSH levels, and an increase in the formation of 4-hydroxynonenal-adduct and 8-hydroxy-2-deoxyguanosine-adduct. Both Nrf2 and NF-κB pathway contributed to the initiation of oxidative stress but the timing of two factors was different. Nrf2 and its related downstream genes were activated earlier than that in NF-κB pathway. In conclusion, AA-induced neurotoxicity attribute to oxidative stress via Nrf2 and NF-κB pathway. Moreover, the co-culture cell model was proven to be a viable model to study AA neurotoxicity.

  11. Cross cultural training in primary mental health care consultations in Moldova - The tEACH perspective.

    PubMed

    Møller, Jane Ege; van Weel-Baumgarten, Evelyn

    2017-09-01

    This article reports experiences and challenges encountered in a cross-cultural training project in Moldova that was undertaken by tEACH, the teaching subcommittee of EACH: International Association for Communication in Healthcare, in cooperation with local and international stakeholders. As part of a major health policy reform, the aim was to equip a group of trainers with the skills to train Moldovan professionals in skills for primary mental health care, including communication skills. The project consisted of 3 weeks of training using mainly experiential teaching methods to allow participants to practice content and methods, including interactive lecturing, roleplay, feedback and video. A majority of the participants reported that they acquired key facilitation skills. They valued the opportunity to practice and receive feedback. However, some reported that there was too much focus on communication skills, which was thought to be less relevant in a Moldovan context. Furthermore our learner-centered approach was occasionally experienced as a lack of structure CONCLUSION: The tEACH expertise plays an important role in supporting trainers in cross-cultural contexts with effective communication skills methods. Teaching in a cross-cultural context is only successful through continuous dialogue with stakeholders and demands attention to cultural differences. Copyright © 2017. Published by Elsevier B.V.

  12. Claudins in a primary cultured puffer fish (Tetraodon nigroviridis) gill epithelium.

    PubMed

    Bui, Phuong; Kelly, Scott P

    2011-01-01

    A primary cultured gill epithelium from the model organism Tetraodon nigroviridis (spotted green puffer fish) has been developed for the study of claudin tight junction (TJ) proteins and their potential role in the regulation of paracellular permeability across the gills of fishes. The cultured preparation is composed of polygonal epithelial cells that exhibit TJ protein immunoreactivity around the periphery and develop a surface morphology of concentric apical microridges. There is an absence of cells exhibiting intense Na+-K+-ATPase immunoreactivity and taken together, these characteristics indicate that the epithelium is composed of gill pavement cells only. In Tetraodon, 52 genes encoding for claudin isoforms (Tncldn) have been identified and 32 of these genes are expressed in whole gill tissue. Of these genes, 12 are responsive to alterations in environmental salinity in vivo (Tncldn3a, -3c, -6, -8d, -10d, -10e, -11a, -23b, -27a, -27c, -32a, and -33b). All claudin isoforms found in whole gill tissue can be found in cultured pavement cell gill epithelia with the exception of Tncldn6, -10d, and -10e. The cultured preparation is suitable for studying the "molecular machinery" of TJ proteins in fish gill pavement cells.

  13. Immobilization of primary cultures of insulin-releasing human pancreatic cells.

    PubMed

    Mantovani, Marluce da Cunha; da Conceição, Mateus Meneghesso; Ferreira, Ari José Scattone; Labriola, Letícia; dos Santos, Patrícia Barros; Tonso, Aldo; Pereira, Carlos Augusto; El-Dorry, Hamza; Sogayar, Mari Cleide

    2009-01-01

    Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type1 Diabetes, however, it is severely limited by the shortage of organ donors. Ex-vivo islet cell cultures appear as an attractive but still elusive approach for curing type 1 Diabetes. It has recently been shown that, even in the absence of fibrotic overgrowth, several factors, such as insufficient nutrition of the islet core, represent a major barrier for long-term survival of islets grafts. The use of immobilized dispersed cells may contribute to solve this problem due to conceivably easier nutritional and oxygen support to the cells.  Therefore, we set out to establish an immobilization method for primary cultures of human pancreatic cells by adsorption onto microcarriers (MCs). Dispersed human islets cells were seeded onto Cytodex1 microcarriers and cultured in bioreactors for up to eight days. The cell number increased and islet cells maintained their insulin secretion levels throughout the time period studied. Moreover, the cells also presented a tendency to cluster upon five days culturing.  Therefore, this procedure represents a useful tool for controlled studies on islet cells physiology and, also, for biotechnological applications.

  14. The Ububele Baby Mat Service - A primary preventative mental health intervention in a culturally diverse setting.

    PubMed

    Dawson, N; Richards, J; Frost, K

    2017-05-01

    The Ububele Baby Mat Service is a community-based, parent-infant mental health intervention offered at five primary health care clinics in Alexandra Township, in Johannesburg. The aim of the intervention is to promote healthy caregiver-infant attachments. There has been a steady increase in the number of mother-baby dyads making use of the service. This paper aims to explore how the Baby Mat Service and Baby Mat practitioners position and locate themselves in a culturally diverse community setting where multiple meaning systems are drawn on for making sense of health concerns. Two key components of the Baby Mat Service are discussed: i) the presence of an inter-racial therapeutic couple on the mat; and ii) the stance adopted by the Baby Mat practitioners in relation to culturally diverse understandings of a presenting problem. The therapeutic couple engages with culturally-informed frames of reference in an attitude of wondering and tentative thinking ("mhlawumbe" in isiZulu). When a respectfully curious stance was taken by practitioners, it was found to help those accessing the mat to find symbolic meaning in the presenting problem and integrate this with cultural understandings.

  15. Analysis of human cytomegalovirus replication in primary cultured human corneal endothelial cells

    PubMed Central

    Hosogai, Mayumi; Shima, Nobuyuki; Nakatani, Yoko; Inoue, Teruki; Iso, Tatsuya; Yokoo, Hideaki; Yorifuji, Hiroshi; Akiyama, Hideo; Kishi, Shoji; Isomura, Hiroki

    2015-01-01

    Background/aims Since the first case of human cytomegalovirus (HCMV)-induced corneal endotheliitis in which HCMV DNA was detected from the patient's aqueous humour using PCR, the clinical evidence for HCMV endotheliitis has been accumulating. However, it remains to be confirmed whether HCMV can efficiently replicate in corneal endothelial cells. We, therefore, sought to determine whether primary cultured human corneal endothelial cells (HCECs) could support HCMV replication. Methods Human foreskin fibroblasts (HFFs) have been shown to be fully permissive for HCMV replication, and are commonly used as an in vitro model for HCMV lytic replication. Therefore, primary cultured HCECs or HFFs were infected with the vascular endotheliotropic HCMV strain TB40/E or laboratory strain Towne. We then compared viral mRNA and protein expression, genome replication and growth between the TB40/E-infected and Towne-infected HCECs and HFFs. Results When HCECs were infected with TB40/E or Towne, rounded cells resembling owl's eyes as well as viral antigens were detected. Viral mRNA synthesis and protein expression proceeded efficiently in the HCECs and HFFs infected with TB40/E or Towne at a high multiplicity of infection (MOI). Similarly, the viral genome was also effectively replicated, with UL44—a viral DNA polymerase processivity factor—foci observed in the nuclei of HCECs. HCECs produced a substantial number of infectious virions after infection with TB40/E at both a high and low MOI. Conclusions Primary cultured HCECs could efficiently support HCMV replication after infection at both a high and low MOI. PMID:26261231

  16. Fibronectin is not Present in the Focal Adhesions Formed between Normal Cultured Fibroblasts and Their Substrata

    NASA Astrophysics Data System (ADS)

    Chen, Wen-Tien; Singer, S. J.

    1980-12-01

    Fibronectin is an extracellular matrix protein that has been implicated in the spreading and adhesion of cultured fibroblasts to their substrata. In this paper, double immunoelectron microscopic labeling experiments for fibronectin and for concanavalin A-binding proteins on the cell surface were carried out on ultrathin frozen sections of cultures of embryonic chicken heart fibroblasts. On cross sections through the focal adhesions of the cell to the substratum there was substantial labeling for concanavalin A-binding proteins but no detectable labeling for fibronectin, whereas both the binding proteins and fibronectin were extensively labeled elsewhere on the cell surface and substratum. These results demonstrate that fibronectin is not present within the sites of focal adhesions. Therefore, the functions of fibronectin in cell spreading and adhesion are not directly mediated through its binding at focal adhesion sites. An alternative model is presented which can account for such fibronectin functions.

  17. Fluorescence-based co-culture of normal and cancerous cells as an indicator of therapeutic effects in cancer.

    PubMed

    Tamura, Masato; Matsui, Hirofumi; Hyodo, Ichinosuke; Tanaka, Junko; Miwa, Yoshihiro

    2014-10-15

    Comprehensive evaluation of the effects of cancer therapies in vitro is difficult because of the need to distinguish the main effects from the side effects within the data. This problem cannot be overcome by methods involving monoculture, because the effects of anti-cancer drugs in a monoculture can only be measured on either normal or cancerous cells in isolation. In order to promote therapeutic development, therefore, we need a novel drug evaluation method which can simultaneously determine both therapeutic activity and toxicity under a co-culture of normal and cancerous cells. Co-culture creates a more biomimetic condition in comparison to monoculture. The novel method proposed in this study uses an easy experiment for estimating the effects of treatments with various kinds of drugs as a solution to the abovementioned problems. We have previously established two cell lines: a rat gastric mucosal cell line (RGM) and its corresponding cancerous mutant cell line (RGK). In this study, we have developed a new evaluation procedure using a co-culture of green fluorescent protein-expressing RGM cells (RGM-GFP) and kusabira orange-expressing RGK cells (RGK-KO). These cell lines emit green and red fluorescence, respectively. We demonstrated the capability of the method in evaluations of the cancer-selective effects of anti-cancer drugs and X-ray treatment. These results clearly distinguished the cancer-selective toxicity of the applied therapies.

  18. Three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells.

    PubMed

    Pal, Anupama; Kleer, Celina G

    2014-04-25

    Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior(1). However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex vivo system(2,3). Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D(4). 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved(3). One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D(6,7). Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during

  19. What pediatricians should know about normal language development: ensuring cultural differences are not diagnosed as disorders.

    PubMed

    Weiss, Amy L; Van Haren, Melissa S

    2003-07-01

    The roles and responsibilities of speech-language pathologists and pediatricians have become greater with the changing population demographics in the United States. In some states, the majority of the population belongs to a national cultural minority, eg, New Mexico. Even a state such as Iowa, with only a 5% nonmajority population, has a school-aged population that is almost 10% nonmajority. This growth of diversity is likely to continue. Rather than viewing sensitivity to the influence of culture on language learning and other developmental areas as an "add-on" to a practice, it may be wiser to recognize that approaching all clients with as few assumptions about their behaviors as possible will guarantee nonbiased service delivery for all. Without nonbiased service delivery, incorrect diagnoses and provision of inappropriate therapy become more likely. Fortunately, many resources are available to assist pediatricians and speech-language pathologists in learning about various cultures. Institutional review boards have become more vigilant about the inclusion of a cross-section of subject populations as participants in research studies in addition to protecting the rights of all participants. Funding agencies also have expressed as a priority the inclusion of research subjects from minority populations to add to the information available about the incidence and prevalence of disorders across the range of our potential patients. In a society in which cultural differences are not just defined by race or ethnicity, but by gender, sexual orientation, age, geographic region, and religion, belief systems about disease, disability, and treatment are dynamic entities for health professionals to take into consideration. It is a challenge that speech-language pathologists and pediatricians must meet if they are to provide the best and most appropriate services for their patients.

  20. Comparison of multiple assays for detecting human antibodies directed against surface antigens on normal and malignant human tissue culture cells.

    PubMed

    Rosenberg, S A; Schwarz, S; Anding, H; Hyatt, C; Williams, G M

    1977-01-01

    Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cell lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual and end-point cytolysis assay and the 51Chromium release assay were equally sensitive in measuring complement mediated antibody cytoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal cells from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody.

  1. Process cost and facility considerations in the selection of primary cell culture clarification technology.

    PubMed

    Felo, Michael; Christensen, Brandon; Higgins, John

    2013-01-01

    The bioreactor volume delineating the selection of primary clarification technology is not always easily defined. Development of a commercial scale process for the manufacture of therapeutic proteins requires scale-up from a few liters to thousands of liters. While the separation techniques used for protein purification are largely conserved across scales, the separation techniques for primary cell culture clarification vary with scale. Process models were developed to compare monoclonal antibody production costs using two cell culture clarification technologies. One process model was created for cell culture clarification by disc stack centrifugation with depth filtration. A second process model was created for clarification by multi-stage depth filtration. Analyses were performed to examine the influence of bioreactor volume, product titer, depth filter capacity, and facility utilization on overall operating costs. At bioreactor volumes <1,000 L, clarification using multi-stage depth filtration offers cost savings compared to clarification using centrifugation. For bioreactor volumes >5,000 L, clarification using centrifugation followed by depth filtration offers significant cost savings. For bioreactor volumes of ∼ 2,000 L, clarification costs are similar between depth filtration and centrifugation. At this scale, factors including facility utilization, available capital, ease of process development, implementation timelines, and process performance characterization play an important role in clarification technology selection. In the case study presented, a multi-product facility selected multi-stage depth filtration for cell culture clarification at the 500 and 2,000 L scales of operation. Facility implementation timelines, process development activities, equipment commissioning and validation, scale-up effects, and process robustness are examined.

  2. Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures.

    PubMed

    Fiorentino, Ilaria; Gualtieri, Roberto; Barbato, Vincenza; Mollo, Valentina; Braun, Sabrina; Angrisani, Alberto; Turano, Mimmo; Furia, Maria; Netti, Paolo A; Guarnieri, Daniela; Fusco, Sabato; Talevi, Riccardo

    2015-01-15

    Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Primary culture and plasmid electroporation of the murine organ of Corti.

    PubMed

    Parker, Mark; Brugeaud, Aurore; Edge, Albert S B

    2010-02-04

    In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells. An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants. This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary

  4. Metabolism and cytotoxic effects of T-2 toxin and its metabolites on human cells in primary culture.

    PubMed

    Königs, Maika; Mulac, Dennis; Schwerdt, Gerald; Gekle, Michael; Humpf, Hans-Ulrich

    2009-04-28

    T-2 toxin belongs to the large group of trichothecene mycotoxins synthesized by various Fusarium molds which can infect raw agriculture materials. Among the trichothecenes, T-2 toxin is one of the most potent mycotoxins and poses a potential health risk in human nutrition. Several acute and chronic toxic effects were observed in humans after consumption of contaminated food. Due to the rapid metabolism of T-2 toxin by esterases, several metabolites can be found in food and also in vivo after ingestion. The aim of this work was to determine the effects of T-2 toxin and of several of its metabolites, namely HT-2 toxin, neosolaniol, T-2-triol and T-2 tetraol, on two human cells in primary culture: human renal proximal tubule epithelial cells (RPTEC) and normal human lung fibroblasts (NHLF). Concerning the cytotoxicity of T-2 toxin and its metabolites, different studies were performed with animal cells and cell lines but there are only little data about cytotoxic effects in human cells. The use of human cells in primary culture gives a good completion of the already known data because these might be limited due to the disadvantages of cell lines (e.g., immortalization, tumor derivation, longtime cultivation). In order to study the cytotoxicity and mode of cell death, the parameters cell viability, caspase-3-activity and LDH-release were measured after exposure to T-2 toxin and several of its metabolites. With IC(50) values of 0.2 and 0.5 microM T-2 toxin showed the strongest cytotoxic effect in both cells with triggering apoptosis as kind of cell death starting at a concentration of 100nM. The metabolites HT-2 toxin and neosolaniol revealed weaker cytotoxic effects (IC(50): 0.7-3.0 microM) and induced apoptosis at higher concentrations (>1 microM). The other metabolites were less cytotoxic (IC(50): 8.3-25.1 microM) and did not activate caspase-3. In addition to the analysis of cytotoxic effects, we also studied the metabolism of T-2 toxin in these cells in primary

  5. Effects of phenytoin on [3H]diazepam binding in dissociated primary cortical cell culture.

    PubMed

    Gallager, D W; Mallorga, P; Swaiman, K F; Neale, E A; Nelson, P G

    1981-08-10

    The effects of chronic exposure of primary dissociated cerebral cortical cells in culture to the anticonvulsant drug phenytoin have been investigated using benzodiazepine binding techniques. By separating benzodiazepine binding into pharmacologically distinct subtypes, the data indicate that clonazepam-displaceable benzodiazepine binding (associated primarily with neuronal membranes) is significantly decreased by exposure to therapeutic and toxic doses of phenytoin while R05-4864-displaceable benzodiazepine binding (associated principally with non-neuronal elements) is enhanced. The ratio of clonazepam-displaceable to R05-4864-displaceable benzodiazepine binding appears to be the most sensitive indicator for these changes.

  6. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures.

    PubMed

    Rao, Vasudev R; Eugenin, Eliseo A; Prasad, Vinayaka R

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood-brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes.

  7. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures

    PubMed Central

    Rao, Vasudev R.; Eugenin, Eliseo A.; Prasad, Vinayaka R.

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood–brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes. PMID:26714725

  8. In vitro culture of oocytes and granulosa cells collected from normal, obese, emaciated and metabolically stressed ewes.

    PubMed

    Tripathi, S K; Farman, M; Nandi, S; Mondal, S; Gupta, Psp; Kumar, V Girish

    2016-07-01

    The present study was undertaken to investigate the oocyte morphology, its fertilizing capacity and granulosa cell functions in ewes (obese, normal, metabolic stressed and emaciated). Ewes (Ovis aries) of approximately 3 years of age (Bellary breed) from a local village were screened, chosen and categorized into a) normal b) obese but not metabolically stressed, c) Emaciated but not metabolically stressed d) Metabolically stressed based on body condition scoring and blood markers. Oocytes and granulosa cells were collected from ovaries of the ewes of all categories after slaughter and were classified into good (oocytes with more than three layers of cumulus cells and homogenous ooplasm), fair (oocytes one or two layers of cumulus cells and homogenous ooplasm) and poor (denuded oocytes or with dark ooplasm). The good and fair quality oocytes were in vitro matured and cultured with fresh semen present and the fertilization, cleavage and blastocyst development were observed. The granulosa cells were cultured for evaluation of metabolic activity by use of the MTT assay, and cell viability, cell number as well as estrogen and progesterone production were assessed. It was observed that the good and fair quality oocytes had greater metabolic activity when collected from normal and obese ewes compared with those from emaciated and metabolically stressed ewes. No significant difference was observed in oocyte quality and maturation amongst the oocytes collected from normal and obese ewes. The cleavage and blastocyst production rates were different for the various body condition classifications and when ranked were: normal>obese>metabolically stressed>emaciated. Lesser metabolic activity was observed in granulosa cells obtained from ovaries of emaciated ewes. However, no changes were observed in viability and cell number of granulosa cells obtained from ewes with the different body condition categories. Estrogen and progesterone production from cultured granulosa cells were

  9. Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures.

    PubMed

    Mammone, Thomas; Gan, David; Foyouzi-Youssefi, Reyhaneh

    2006-11-01

    Normal human dermal fibroblasts have a limited life-span in vitro and stop proliferation after a fixed number of cell divisions. This process by which cells stop proliferation is called senescence. Senescence is also characterized by a decrease in the total cell number. In this study, we characterized an increase in cell death in normal human dermal fibroblasts in vitro as a function of increasing cell passage. With increasing passage, human fibroblasts showed an increase in the number of dead cells and increased DNA fragmentation as determined by flow cytometry. Serial passage of human fibroblasts also resulted in mitochondrial dysfunction, represented by a loss of mitochondrial membrane potential. The apoptotic markers caspase-3 and cytochrome c were both found to increase in senescent cells. These results suggest the activation of an apoptotic pathway within a population of human fibroblasts as a function of cell passage.

  10. Air pollutants cause release of hydrogen peroxide and interleukin-8 in a human primary nasal tissue culture model.

    PubMed

    Cho, Do-Yeon; Le, Wei; Bravo, Dawn T; Hwang, Peter H; Illek, Beate; Fischer, Horst; Nayak, Jayakar V

    2014-12-01

    A component of primary innate defense of the nasal mucosa against inhaled pathogens includes continuous, low-level release of hydrogen peroxide (H2 O2 ) into luminal secretions. Epidemiologically, an association exists between poor air quality and increased prevalence of sinonasal disease. To understand the effects of particulate matter (PM) in nasal mucosa, we studied the release of H2 O2 and interleukin 8 (IL-8) after PM exposure. Human nasal specimens were collected from surgery and cultured in serum-free growth medium. Cell integrity and recovery during culture was monitored by lactate dehydrogenase (LDH) release into the medium. Cultures were exposed to PM for 24 hours in the presence/absence of diphenyleneiodonium sulfate (DPI; a nicotinamide adenine dinucleotide phosphate [NADPH] oxidase inhibitor). Luminex cytokine and Amplex-Red H2 O2 assays were performed. LDH levels dropped rapidly within 2 days, indicative of stabilization and cell recovery after harvest. All cultures released H2 O2 into the medium. Exposure to PM (20 μg/cm(2) ) increased H2 O2 levels significantly (94.6 ± 7.7 nM) compared to untreated controls (55.8 ± 4.0 nM; p = 0.001). PM-induced H2 O2 production was partially inhibited by DPI (80.1 ± 3.8nM), indicating that cellular NADPH oxidase may be a primary source of H2 O2 production. Exposure to PM increased IL-8 levels in a dose-dependent fashion (control = 2301 ± 412 MFI; 20 μg/cm(2) = 5002 ± 1327 MFI; 40 μg/cm(2) = 8219 ± 1090 MFI; p = 0.022). PM increases the quantity of H2 O2 released by nasal epithelial cells, indicating that PM can contribute to oxidative stress in part by activating a normal cellular defense mechanism. Exposure to PM resulted in elevated IL-8 levels and mucin production in explants. Efforts to reduce airborne PM may lead to reduced H2 O2 and mucin production in sinonasal epithelium. © 2014 ARS-AAOA, LLC.

  11. Cytotoxicity, metabolism and cellular uptake of the mycotoxin deoxynivalenol in human proximal tubule cells and lung fibroblasts in primary culture.

    PubMed

    Königs, Maika; Lenczyk, Marlies; Schwerdt, Gerald; Holzinger, Hildegard; Gekle, Michael; Humpf, Hans-Ulrich

    2007-10-30

    At the level of the whole animal, the toxic effects of the mycotoxin deoxynivalenol (DON) range from causing diarrhoea, vomiting, gastro-intestinal inflammation to necrosis of several tissues. It also affects the immune system and leads to kidney lesions. Although DON has been tested in different human and animal cell lines for its cytotoxicity, these tests might be limited due to the disadvantages of cell lines (e.g. immortalization, tumour derivation, longtime cultivation) and do not necessarily reflect the response of normal cells. In order to overcome this problem and to be closer to the human situation, we studied the effect of DON in human kidney epithelial cells (renal proximal tubule epithelial cells, RPTEC) and human lung fibroblasts (normal human lung fibroblast, NHLF) in primary culture. Cell viability, apoptotic and necrotic cell death, collagens I, III and IV as well as fibronectin secretion were determined. It could be demonstrated that DON has a distinct cytotoxic effect on human primary cells. A reduction in viability can be observed in both cell types, with fibroblasts reacting more sensitive. Furthermore, DON caused mainly necrotic cell death in kidney cells whereas mainly apoptotic cell death in fibroblasts. DON had no effect on collagen secretion in RPTEC cells. Collagen secretion was partially decreased in NHLF. In both cells, fibronectin secretion was reduced after 5 days of exposure. We also studied the metabolism and the cellular uptake of DON using LC-MS/MS. DON was neither metabolized by proximal tubule cells nor by fibroblasts. DON is incorporated into the cells whereas the intracellular amount of DON in kidney cells is higher than in fibroblasts. No accumulation of DON occurred in the cells.

  12. Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures.

    PubMed

    Petrenko, Volodymyr; Saini, Camille; Perrin, Laurent; Dibner, Charna

    2016-11-11

    Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

  13. Effects of methylmercury on primary cultured rat hepatocytes: Cell injury and inhibition of growth factor stimulated DNA synthesis

    SciTech Connect

    Tanno, Keiichi; Fukazawa, Toshiyuki; Tajima, Shizuko; Fujiki, Motoo )

    1992-08-01

    Many more studies deal with the toxicity of methylmercury on nervous tissue than on its toxicity to the liver. Methylmercury accumulates in the liver in higher concentrations than brain and the liver has the primary function of detoxifying methylmercury. According to recent studies, hepatocyte mitochondrial membranes are destroyed by methylmercury and DNA synthesis is inhibited by methylmercury during hepatocyte regeneration. Methylmercury alters the membrane ion permeability of isolate skate hepatocytes, and inhibits the metal-sensitive alcohol dehydrogenase and glutathione reductase of primary cultured rat hepatocytes. However, little is known about the effect of methylmercury on hepatocyte proliferation in primary cultured rat hepatocytes. We therefore used the primary cultured rat hepatocytes to investigate the effects of methylmercury on cell injury and growth factor stimulate DNA synthesis. The primary effect of methylmercury is to inhibit hepatocyte proliferation rather than to cause direct cell injury. 16 refs., 4 figs.

  14. Analysis of soluble factors in conditioned media derived from primary cultures of cirrhotic liver of biliary atresia.

    PubMed

    Yamazaki, Taisuke; Wakai, Mariko; Enosawa, Shin; Tokiwa, Takayoshi

    2017-06-01

    Biliary atresia (BA) is a rare and serious liver disease in newborn infants. Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of BA may contain hepatic stem/progenitor cells in primary culture of NPC fractions. In this study, NPC fractions were subjected to primary or passage culture and found that clusters of hepatocyte-like cells appear even without adding hepatocyte growth factor (HGF) to the culture medium, but not in their passage culture used as a control. Based on these findings, conditioned media (CMs) were collected and soluble factors in the CMs were analyzed in order to elucidate the mechanism of the appearance of hepatocyte-like cells or their clusters. A large amount of active HGF consisting of α and β chains was detected in CMs derived from primary culture, but not in CMs from passage culture, as determined by western blot analysis, bone morphogenetic protein (BMP)-4, oncostatin M (OSM), and transforming growth factor (TGF)-β1 were not detected in any of the CMs. The number of hepatocyte-like cells in primary culture tended to decrease following treatment with the HGF receptor c-Met inhibitor, SU11274 in a dose-dependent manner. Furthermore, the clusters of hepatocyte-like cells tended to increase in size and number when freshly isolated NPC fractions were cultured in the presence of 10% of CMs collected after 3-4 wk of primary culture. In conclusion, these findings indicate that CMs derived from primary culture of NPC fractions of BA liver contain a large amount of active HGF, which may activate hepatic stem/progenitor cells and promote the appearance of hepatocyte-like cells or their clusters through HGF/c-Met signaling. The present study would lead to cell therapy using the patient's own cells for the treatment of BA.

  15. Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats.

    PubMed

    Richert, Lysiane; Baze, Audrey; Parmentier, Céline; Gerets, Helga H J; Sison-Young, Rowena; Dorau, Martina; Lovatt, Cerys; Czich, Andreas; Goldring, Christopher; Park, B Kevin; Juhila, Satu; Foster, Alison J; Williams, Dominic P

    2016-09-06

    Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug.

  16. Ultrastructural characterization of normal and abnormal chondrogenesis in micromass rat embryo limb bud cell cultures.

    PubMed

    Renault, J Y; Caillaud, J M; Chevalier, J

    1995-02-01

    Inhibition of chondrogenesis in limb bud cell micromass cultures has been proposed as a short-term teratogen detection test. Validation studies were performed by testing large series of reference compounds and comparing their teratogenic potential with their ability to inhibit chondrogenesis; however, there are few reports describing the histological and ultrastructural changes associated with inhibition of chondrogenesis in vitro. The objective of this study was to provide a qualitative description of the histological and ultrastructural alterations induced by three chondrogenesis inhibitors: retinoic acid (RA) and 6-aminonicotinamide (6AN), two teratogens, and doxylamine succinate (DS), a nonteratogen compound. In addition, in order to have a basis for the interpretation of the morphological alterations induced by the test compounds, the histological and ultrastructural changes which occur during the time course of chondrogenesis in control cultures were described and compared with those in rat embryo limb buds. We found that RA at 0.5 micrograms/ml led to a marked decrease in the number and size of cartilaginous foci; most cells lacked morphological signs of differentiation but their ability to proliferate was unaffected. At concentrations of 2 micrograms/ml and more, 6AN delayed cell proliferation, reduced staining of the extracellular matrix, and induced the formation of endoplasmic cisternae. DS at 50 micrograms/ml affected both differentiation and proliferation; pigment deposits were observed in chondrocytes, suggesting phospholipid metabolism disorders. In conclusion, this study showed that inhibition of chondrogenesis in this simple cell culture system can be associated with different types of histological and ultrastructural alterations. Examination of these alterations can provide useful information about the teratogenic potential of tested compounds and their mechanism of action.

  17. Nontargeted Stressful Effects in Normal Human Fibroblast Cultures Exposed to Low Fluences of High Charge, High Energy (HZE) Particles: Kinetics of Biologic Responses and Significance of Secondary Radiations

    PubMed Central

    Gonon, Géraldine; Groetz, Jean-Emmanuel; de Toledo, Sonia M.; Howell, Roger W.; Fromm, Michel; Azzam, Edouard I.

    2014-01-01

    The induction of nontargeted stressful effects in cell populations exposed to low fluences of high charge (Z) and high energy (E) particles is relevant to estimates of the health risks of space radiation. We investigated the up-regulation of stress markers in confluent normal human fibroblast cultures exposed to 1,000 MeV/u iron ions [linear energy transfer (LET) ~151 keV/μm] or 600 MeV/u silicon ions (LET ~50 keV/μm) at mean absorbed doses as low as 0.2 cGy, wherein 1–3% of the cells were targeted through the nucleus by a primary particle. Within 24 h postirradiation, significant increases in the levels of phospho-TP53 (serine 15), p21Waf1 (CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation were detected, which suggested participation in the stress response of cells not targeted by primary particles. This was supported by in situ studies that indicated greater increases in 53BP1 foci formation, a marker of DNA damage. than expected from the number of primary particle traversals. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure of the cell cultures to 0.2 cGy of 3.7 MeV α particles (LET ~109 keV/μm) that targets ~1.6% of nuclei, but not after 0.2 cGy from 290 MeV/u carbon ions (LET ~13 keV/μm) by which, on average, ~13% of the nuclei were hit, which highlights the importance of radiation quality in the induced effect. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cell cultures exposed to HZE particles comprise <1% of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10–20 μm. Thus, the latter are unlikely to significantly contribute to stressful effects in cells not targeted by primary HZE particles. PMID:23465079

  18. Putative nanobacteria represent physiological remnants and culture by-products of normal calcium homeostasis.

    PubMed

    Young, John D; Martel, Jan; Young, Lena; Wu, Cheng-Yeu; Young, Andrew; Young, David

    2009-01-01

    Putative living entities called nanobacteria (NB) are unusual for their small sizes (50-500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures described

  19. Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

    PubMed Central

    Young, John D.; Young, Lena; Wu, Cheng-Yeu; Young, Andrew

    2009-01-01

    Putative living entities called nanobacteria (NB) are unusual for their small sizes (50–500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures

  20. Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

    EPA Science Inventory

    Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the ...

  1. The Influence of Cultural Bias on Motivation to Learn English: The Case of Khoe Primary School Students in Eastern Botswana

    ERIC Educational Resources Information Center

    Magogwe, Joel Mokuedi

    2009-01-01

    This study investigated the influence of cultural bias in the teaching of English and in the books used to teach English in primary schools attended by Khoe students in eastern Botswana. The study also explored the link between cultural bias and the attitudes and motivation of Khoe students learning English. One hundred and thirty-seven students…

  2. Chinese Cultural Education in Post-Colonial Hong Kong: Primary School Chinese Language Teachers' Belief and Practice

    ERIC Educational Resources Information Center

    Kwan, Ming Kai Marko

    2010-01-01

    Before 1997, no formal curriculum on Chinese cultural education for primary schools was developed in Hong Kong although the education authority had started to introduce some items of Chinese cultural learning into the Chinese language syllabus when the Target Oriented Curriculum was implemented in 1996. However, such items were incorporated into…

  3. Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

    EPA Science Inventory

    Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the ...

  4. Chinese Cultural Education in Post-Colonial Hong Kong: Primary School Chinese Language Teachers' Belief and Practice

    ERIC Educational Resources Information Center

    Kwan, Ming Kai Marko

    2010-01-01

    Before 1997, no formal curriculum on Chinese cultural education for primary schools was developed in Hong Kong although the education authority had started to introduce some items of Chinese cultural learning into the Chinese language syllabus when the Target Oriented Curriculum was implemented in 1996. However, such items were incorporated into…

  5. Mitochondrial activity assessed by cytofluorescence after in-vitro-irradiation of primary rat brain cultures

    SciTech Connect

    Cervos-Navarro, J.; Hamdorf, G. )

    1993-05-01

    Mitochondria play a key role in cell homeostasis and are the first cell organells affected by ionizing irradiation, as it was proved by previous electron microscopic investigations. In order to observe functional parameters of mitochondria after low-dose irradiation, primary rat brain cultures (prepared from 15-day-old rat fetuses) were irradiated from a [sup 60]Co-source with 0.5 and 1 Gy at the age of 2 or 7 days in vitro (div). Cytofluorescence measurement was made by a Cytofluor[sup [trademark]2350] using Rhodamine 123. This fluorescent dye is positively charged and accumulates specifically in the mitochondria of living cells without cytotoxic effect. Since its retention depends on the negative membrane potential as well as the proton gradient that exists across the inner mitochondrial membrane, Rhodamine 123 accumulation reflects the status of mitochondrial activity as a whole. After irradiation with 0.5 and 1 Gy on day 2 in culture there was a decrease in Rhodamine uptake in the irradiated cultures during the first week after the irradiation insult which reached minimum values after 3 days. Rhodamine uptake increased during the following period and finally reached the values of the control cultures. In the second experiment with irradiated cultures on day 7 and the same doses of 0.5 and 1 Gy the accumulation of Rhodamine decreased only initially then increased tremendously. After both doses values of Rhodamine-accumulation were higher than the control level. The results demonstrated that irradiation caused a change in mitochondrial activity depending on the time of irradiation. The dramatic increase over the control levels after irradiation on day 7 in vitro is attributed to the fact that at this time synapses have already developed. Deficiency of mitochondrial activity as well as hyperactivity and the consequent change in energy production may lead to changes in neuronal metabolism including an increase in production of free radicals.

  6. Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes.

    PubMed

    Savoian, Matthew S

    2015-07-01

    In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems.

  7. Influence of workplace culture on nursing-sensitive nurse outcomes in municipal primary health care.

    PubMed

    Hahtela, Nina; Paavilainen, Eija; McCormack, Brendan; Slater, Paul; Helminen, Mika; Suominen, Tarja

    2015-10-01

    To explore the influence of workplace culture on sickness absences, overtime work and occupational injuries in municipal primary health care. The need to improve nursing sensitive outcomes has been highlighted. Therefore, an adequate understanding of the influence of workplace culture on nursing-sensitive nurse outcomes is essential for nurse managers to meet the requirements of improving nursing outcomes. A cross-sectional survey design was used to incorporating the data from 21 inpatient acute care units of nine organisations at the Finnish municipal primary health care system from 2011 to 2012. Findings emphasise in particular the importance of the practice environment as being an interpretative factor for nurses' absences owing to sickness, overtime work and occupational injuries. To ensure favourable nursing sensitive outcomes it is essential that there is a shared interest in the unit to invest in the creation of a supportive practice environment. Outcome improvements require a special focus on issues related to nursing management, adequate staffing and resources and intention to leave. © 2014 John Wiley & Sons Ltd.

  8. Replication of human immunodeficiency virus type 1 in primary dendritic cell cultures.

    PubMed Central

    Langhoff, E; Terwilliger, E F; Bos, H J; Kalland, K H; Poznansky, M C; Bacon, O M; Haseltine, W A

    1991-01-01

    The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in primary blood dendritic cells was investigated. Dendritic cells compose less than 1% of the circulating leukocytes and are nondividing cells. Highly purified preparations of dendritic cells were obtained using recent advances in cell fractionation. The results of these experiments show that dendritic cells, in contrast to monocytes and T cells, support the active replication of all strains of HIV-1 tested, including T-cell tropic and monocyte/macrophage tropic isolates. The dendritic cell cultures supported much more virus production than did cultures of primary unseparated T cells, CD4+ T cells, and adherent as well as nonadherent monocytes. Replication of HIV-1 in dendritic cells produces no noticeable cytopathic effect nor does it decrease total cell number. The ability of the nonreplicating dendritic cells to support high levels of replication of HIV-1 suggests that this antigen-presenting cell population, which is also capable of supporting clonal T-cell growth, may play a central role in HIV pathogenesis, serving as a source of continued infection of CD4+ T cells and as a reservoir of virus infection. Images PMID:1910172

  9. Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes

    PubMed Central

    2015-01-01

    In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems. PMID:25802491

  10. Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes.

    PubMed

    Hwang, Geun Hye; Jeon, Yu Jin; Han, Ho Jae; Park, Soo Hyun; Baek, Kyoung Min; Chang, Woochul; Kim, Joong Sun; Kim, Lark Kyun; Lee, You-Mie; Lee, Sangkyu; Bae, Jong-Sup; Jee, Jun-Goo; Lee, Min Young

    2015-01-01

    Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H2O2)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H2O2 in a dose-dependent manner. Additionally, H2O2 treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H2O2 significantly attenuated the H2O2-induced decrease of cell viability. H2O2 exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H2O2-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H2O2-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H2O2-induced apoptosis by inhibiting ROS generation.

  11. Antioxidant lignans from Machilus thunbergii protect CCl4-injured primary cultures of rat hepatocytes.

    PubMed

    Yu, Y U; Kang, S Y; Park, H Y; Sung, S H; Lee, E J; Kim, S Y; Kim, Y C

    2000-09-01

    Eleven lignans (1-11) were isolated from the CH2Cl2 fraction of the bark of Machilus thunbergii Sieb. et Zucc. (Lauraceae). These were identified as (-)-acuminatin (1), (-)-isoguaiacin (2), meso-dihydroguaiaretic acid (3), (+)-galbacin (4), (-)-sesamin (5), (+)-galbelgin (6), machilin A (7), machilin G (8), licarin A (9), and nectandrin A (10) and B (11). Primary cultures of rat hepatocytes were co-incubated for 90 min with the hepatotoxin CCl4 and each of the 11 lignans (50 microM). Hepatoprotective activity was determined by measuring the level of glutamic pyruvic transaminase released into the medium from the primary cultures of rat hepatocytes. (-)-Acuminatin, (-)-isoguaiacin and meso-dihydroguaiaretic acid all significantly reduced the level of glutamic pyruvic transaminase released. Further investigation revealed that these three compounds significantly preserved the levels and the activities of glutathione, superoxide dismutase, glutathione peroxidase and catalase. (-)-Acuminatin, (-)-isoguaiacin and meso-dihydroguaiaretic acid also ameliorated lipid peroxidation as demonstrated by a reduction of malondialdehyde production. These results suggest that (-)-acuminatin, (-)-isoguaiacin and meso-dihydroguaiaretic acid exert diverse hepatoprotective activities, perhaps by serving as potent antioxidants.

  12. Exposure to 1800 MHz radiofrequency radiation induces oxidative damage to mitochondrial DNA in primary cultured neurons.

    PubMed

    Xu, Shangcheng; Zhou, Zhou; Zhang, Lei; Yu, Zhengping; Zhang, Wei; Wang, Yuan; Wang, Xubu; Li, Maoquan; Chen, Yang; Chen, Chunhai; He, Mindi; Zhang, Guangbin; Zhong, Min

    2010-01-22

    Increasing evidence indicates that oxidative stress may be involved in the adverse effects of radiofrequency (RF) radiation on the brain. Because mitochondrial DNA (mtDNA) defects are closely associated with various nervous system diseases and mtDNA is particularly susceptible to oxidative stress, the purpose of this study was to determine whether radiofrequency radiation can cause oxidative damage to mtDNA. In this study, we exposed primary cultured cortical neurons to pulsed RF electromagnetic fields at a frequency of 1800 MHz modulated by 217 Hz at an average special absorption rate (SAR) of 2 W/kg. At 24 h after exposure, we found that RF radiation induced a significant increase in the levels of 8-hydroxyguanine (8-OHdG), a common biomarker of DNA oxidative damage, in the mitochondria of neurons. Concomitant with this finding, the copy number of mtDNA and the levels of mitochondrial RNA (mtRNA) transcripts showed an obvious reduction after RF exposure. Each of these mtDNA disturbances could be reversed by pretreatment with melatonin, which is known to be an efficient antioxidant in the brain. Together, these results suggested that 1800 MHz RF radiation could cause oxidative damage to mtDNA in primary cultured neurons. Oxidative damage to mtDNA may account for the neurotoxicity of RF radiation in the brain.

  13. Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes

    PubMed Central

    Hwang, Geun Hye; Jeon, Yu Jin; Han, Ho Jae; Park, Soo Hyun; Baek, Kyoung Min; Chang, Woochul; Kim, Joong Sun; Kim, Lark Kyun; Lee, You-Mie; Lee, Sangkyu; Bae, Jong-Sup; Jee, Jun-Goo

    2015-01-01

    Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H2O2)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H2O2 in a dose-dependent manner. Additionally, H2O2 treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H2O2 significantly attenuated the H2O2-induced decrease of cell viability. H2O2 exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H2O2-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H2O2-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H2O2-induced apoptosis by inhibiting ROS generation. PMID:25798044

  14. Pyrrolizidine alkaloid clivorine induced oxidative injury on primary cultured rat hepatocytes.

    PubMed

    Ji, LiLi; Liu, TianYu; Wang, ZhengTao

    2010-04-01

    Clivorine is an otonecine-type hepatotoxic pyrrolizidine alkaloid (HPAs), to which humans are exposed when consuming herbs containing such components. In the present study, we investigated clivorine-induced oxidative stress injury on primary cultured rat hepatocytes. Rat hepatocytes were treated with various concentrations of clivorine (1-100 microM) for 48 hours, and then cell viability was detected by 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay, while lipid peroxidation (LPO) level, glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) activities were determined to evaluate the oxidative injury. The results of MTT assay showed that clivorine decreased cell viability in a concentration-dependent manner. Clivorine also increased LPO amounts in rat hepatocytes at the concentrations of 50 microM and 100 microM. Further results showed that clivorine decreased GPx, GST and GR activities, which are all reduced glutathione (GSH)-related antioxidant enzymes. CAT and SOD are both important antioxidant enzymes, and the results showed that clivorine increased CAT activity at the low concentration of 5 muM and decreased cellular SOD activity at all concentrations. Taken together, our results demonstrated that clivorine induced toxicity on primary cultured rat hepatocytes by causing the damage on cellular redox balance.

  15. Evaluation of cytokine gene expression after avian influenza virus infection in avian cell lines and primary cell cultures

    USDA-ARS?s Scientific Manuscript database

    The innate immune responses elicited by avian influenza virus (AIV) infection has been studied by measuring cytokine gene expression by relative real time PCR (rRT-PCR) in vitro, using both cell lines and primary cell cultures. Continuous cell lines offer advantages over the use of primary cell cult...

  16. Melanogenesis and antioxidant defense system in normal human melanocytes cultured in the presence of chlorpromazine.

    PubMed

    Otreba, Michał; Wrześniok, Dorota; Beberok, Artur; Rok, Jakub; Buszman, Ewa

    2015-02-01

    Chlorpromazine is used in the treatment of schizophrenia and psychotic disorders and belongs to phenothiazine class of neuroleptic drugs. It shows severe side effects such as extrapyramidal symptoms as well as ocular and skin disorders, but the mechanism is still not fully established. The aim of this study was to examine the effect of chlorpromazine on cell viability, melanogenesis and antioxidant defense system in normal human melanocytes. It has been demonstrated that chlorpromazine induces concentration dependent loss in cell viability. The value of EC(50) was calculated to be 2.53 μM. Chlorpromazine in lower concentrations (0.0001, 0.001 and 0.01 μM) increased the melanin and microphthalmia-associated transcription factor (MITF) content and tyrosinase activity, while changes of antioxidant enzymes activity were not observed. It suggests that long-term chlorpromazine therapy, even with low drug doses, may lead to hyperpigmentation disorders in skin and/or eye. The use of the analyzed drug in higher concentrations (0.1 and 1.0 μM) caused significant alterations of antioxidant enzymes activity in normal melanocytes, what may explain a potential role of chlorpromazine in the depletion of cellular antioxidant status leading to other adverse effects associated with the high-dose and/or long-term therapy.

  17. Tissue culture of normal and cystic fibrosis sweat gland duct cells. I. Alterations in dome formation.

    PubMed

    Hazen-Martin, D J; Spicer, S S; Sens, M A; Jenkins, M Q; Westphal, M C; Sens, D A

    1987-01-01

    The elucidation of the underlying defect in fluid secretion by cystic fibrosis (CF) sweat glands is hindered by the unavailability of an experimental model for investigating this disease. As a potential model system, a serum-free growth medium was developed that supports the explant growth of epithelial cells from fragments of human skin. Immunohistochemical analysis demonstrated that these epithelial cell outgrowths originated from the duct of the sweat gland. By electron microscopy, the cells were demonstrated to possess keratinocyte-like morphology as noted by the presence of a multilayered outgrowth of cells containing well-defined keratin bundles. Identical outgrowths from skin biopsies of CF patients were compared to normal outgrowths and alterations were noted to occur in dome formation and in the number of intercellular spaces between cells. Doming alterations were also noted to occur in the CF heterozygous state. No differences in cell fine structure or in growth factor requirements for cell proliferation were noted between normal and CF cells. The potential use of this system as a model for CF research is discussed.

  18. The SCOPE-PC instrument for assessing patient safety culture in primary care: a psychometric evaluation.

    PubMed

    Desmedt, Melissa; Bergs, Jochen; Willaert, Benjamin; Vlayen, Annemie; Hellings, Johan; Schrooten, Ward; Claes, Neree; Vandijck, Dominique

    2017-07-09

    Primary healthcare differs from hospitals in terms of - inter alia - organisational structure. Therefore, patient safety culture could differ between these settings. Various instruments have been developed to measure collective attitudes of personnel within a primary healthcare organisation. However, the number of valid and reliable instruments is limited. Psychometric properties of the SCOPE-Primary Care instrument were tested to examine the instrument's applicability in home care services in Belgium. A cross-sectional study was conducted by administering the SCOPE-PC questionnaire in a single home care organisation with more than 1000 employees, including nurses, midwives, healthcare assistants, diabetes educators and nursing supervisors. First, a confirmatory factor analysis was performed to test whether the observed dataset fitted to the proposed seven-factor model of the SCOPE-PC instrument. Second, Cronbach's alphas were calculated to examine internal consistency reliability. Finally, the instrument's validity was also examined. In total, 603 questionnaires were retained for further analysis, representing an overall response rate of 43.9%. Most respondents were nursing staff, followed by healthcare assistants and nursing supervisors. The results of the confirmatory factor analyses satisfied the chosen cut-offs, indicating an acceptable to good model fit. With the exception of the dimension 'organizational learning' (0.58), Cronbach's alpha scores of the SCOPE-PC scales indicated a good level of internal consistency: 'open communication and learning from error' (0.86), 'handover and teamwork' (0.78), 'adequate procedures and working conditions' (0.73), 'patient safety management' (0.81), 'support and fellowship' (0.75), and 'intention to report events (0.85). Moreover, inter-correlations between the seven dimensions as well as with the patient safety grade were moderate to good. The present study indicated that the SCOPE-Primary Care instrument has good

  19. Evaluation of MENT on primary cell cultures from benign prostatic hyperplasia and prostate carcinoma.

    PubMed

    Mendoza, Patricia; Sánchez, Catherine; Contreras, Héctor R; Vergara, Jorge; Acevedo, Cristian; Cabezas, Juan; Huidobro, Christian; Noé, Gabriela; Castellón, Enrique A

    2009-12-01

    7-alpha-Methyl-19-Nortestosterone (MENT) is a synthetic androgen more potent than testosterone (T) and cannot be reduced at 5-alpha position. No important effects of MENT on prostate growth have been reported. However, little is known about the effect of MENT on benign prostatic hyperplasia (BPH) or prostate carcinoma (CaP). We evaluate the effect of MENT, T and dihydrotestosterone (DHT) on secretion, proliferation and gene expression of primary cell cultures from human BPH and CaP. Moreover, the effect of these androgens was examined in the presence of finasteride to determine the influence of the 5-alpha reductase (5-AR) activity on the androgenic potency. BPH and CaP primary cultures were treated with 0, 1, 10 and 100 nM of T, MENT or DHT during 24 and 48 h. Prostate-specific antigen (PSA) was measured by micro particles immunoassay and proliferation rate by spectrophotometric assay (MTT) and by the immunochemical detection of the proliferation marker Ki-67. Gene expression of FGF8b (androgen sensitive gene) was evaluated by semi-quantitative RT-PCR. Results showed that MENT treatments increased PSA secretion and proliferation rate with a potency ranged between T and DHT. Similar effects of MENT were observed in both BPH and CaP cultures. The studies with finasteride showed that in BPH and CaP cells, the conversion of T into DHT significantly contributes to its effect on the proliferation and PSA secretion, and corroborated the resistance of MENT to the 5-AR. The effect of MENT on the gene expression of FGF8b in CaP cells was similar to T and lower than DHT. It is concluded that MENT increases proliferative and secretory activities and gene expression on pathological prostate cells although in less extent than the active metabolite DHT. Furthermore, the fall of endogenous concentration of T during MENT treatment anticipates that this androgen will be of low impact for the prostate.

  20. Characterization of extracellular matrix macromolecules in primary cultures of equine keratinocytes.

    PubMed

    Visser, Michelle B; Pollitt, Christopher C

    2010-03-15

    Most research to date involving laminins and extracellular matrix protein function in both normal and pathological conditions involves in vitro culture of keratinocytes. Few methods are established to allow for prolonged propagation of keratinocytes from equine tissues, including the hoof lamellae. In this study we modified cell isolation and culture techniques to allow for proliferation and sub-culturing of equine lamellar keratinocytes. Additionally, the production and processing of extracellular matrix molecules by skin and lamellar keratinocytes were studied. Physical and proteolytic tissue separation in combination with media containing a calcium concentration of 0.6 mM in combination with additional media supplements proved optimal for proliferation and subculture of equine lamellar keratinocytes on collagen coated substratum. Immunofluorescence and immunoblotting studies confirmed that equine skin and lamellar keratinocytes produce Ln-332 in vitro and processing of this molecule follows that of other species. As well, matrix components including integrin alpha-6 (alpha 6) and the hemidesmosome proteins, bullous pemphigoid antigen 1 (BP180) bullous pemphigoid antigen 2 (BP230) and plectin are also expressed. Isolation of equine keratinocytes and study of the matrix and adhesion related molecules produced by them provides a valuable tool for future work in the veterinary field.

  1. Establishment, characterization, and toxicological application of loggerhead sea turtle (Caretta caretta) primary skin fibroblast cell cultures.

    PubMed

    Webb, Sarah J; Zychowski, Gregory V; Bauman, Sandy W; Higgins, Benjamin M; Raudsepp, Terje; Gollahon, Lauren S; Wooten, Kimberly J; Cole, Jennifer M; Godard-Codding, Céline

    2014-12-16

    Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 μM following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 μM benzo[a]pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells.

  2. An imbalance in progenitor cell populations reflects tumour progression in breast cancer primary culture models

    PubMed Central

    2011-01-01

    Background Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression. Methods Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively. Results Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumourogenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells. Conclusions Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression. PMID:21521500

  3. Expression of adipocyte biomarkers in a primary cell culture models reflects preweaning adipobiology.

    PubMed

    Chu, Dinh-Toi; Malinowska, Elzbieta; Gawronska-Kozak, Barbara; Kozak, Leslie P

    2014-06-27

    A cohort of genes was selected to characterize the adipogenic phenotype in primary cell cultures from three tissue sources. We compared the quantitative expression of biomarkers in culture relative to their expression in vivo because the mere presence or absence of expression is minimally informative. Although all biomarkers analyzed have biochemical functions in adipocytes, the expression of some of the biomarkers varied enormously in culture relative to their expression in the adult fat tissues in vivo, i.e. inguinal fat for white adipocytes and brite cells, interscapular brown adipose tissue for brown adipocytes, and ear mesenchymal stem cells for white adipocytes from adult mice. We propose that the pattern of expression in vitro does not reflect gene expression in the adult mouse; rather it is predominantly the expression pattern of adipose tissue of the developing mouse between birth and weaning. The variation in gene expression among fat depots in both human and rodent has been an extensively studied phenomenon, and as recently reviewed, it is related to subphenotypes associated with immune function, the inflammatory response, fat depot blood flow, and insulin sensitivity. We suggest that adipose tissue biology in the period from birth to weaning is not just a staging platform for the emergence of adult white fat but that it has properties to serve the unique needs of energy metabolism in the newborn. A case in point is the differentiation of brite cells that occurs during this period followed by their involution immediately following weaning.

  4. U. v. -enhanced reactivation of u. v. -irradiated herpes virus by primary cultures of rat hepatocytes

    SciTech Connect

    Zurlo, J.; Yager, J.D. )

    1984-04-01

    Carcinogen treatment of cultured mammalian cells prior to infection with u.v.-irradiated virus results in enhanced virus survival and mutagenesis suggesting the induction of SOS-type processes. The development of a primary rat hepatocyte culture system is reported to investigate cellular responses to DNA damage which may be relevant to hepatocarcinogenesis in vivo. Enhanced reactivation of u.v.-irradiated Herpes simplex virus type 1 (HSV-1) occurred in hepatocytes irradiated with u.v. Cultured hepatocytes were pretreated with u.v. at the time of enhanced DNA synthesis. These treatments caused an inhibition followed by a recovery of DNA synthesis. At various times after pretreatment, the hepatocytes were infected with control or u.v.-irradiated HSV-1 at low multiplicity, and virus survival was measured. U.v.-irradiated HSV-1 exhibited the expected two-component survival curve in control or u.v. pretreated hepatocytes. The magnitude of enhanced reactivation of HSV-1 was dependent on the u.v. dose to the hepatocytes, the time of infection following u.v. pretreatment, and the level of DNA synthesis at the time of pretreatment. These results suggest that u.v. treatment of rat hepatocytes causes the induction of SOS-type functions tht may have a role in the initiation of hepatocarcinogenesis.

  5. Activity of Protease-Activated Receptors in Primary Cultured Human Myenteric Neurons

    PubMed Central

    Kugler, Eva M.; Mazzuoli, Gemma; Demir, Ihsan E.; Ceyhan, Güralp O.; Zeller, Florian; Schemann, Michael

    2012-01-01

    Activity of the four known protease-activated receptors (PARs) has been well studied in rodent enteric nervous system and results in animal models established an important role for neuronal PAR2. We recently demonstrated that, unlike in rodents, PAR1 is the dominant neuronal protease receptor in the human submucous plexus. With this study we investigated whether this also applies to the human myenteric plexus. We used voltage sensitive dye recordings to detect action potential discharge in primary cultures of human myenteric neurons in response to PAR activating peptides (APs). Application of the PAR1-AP (TFLLR) or PAR4-AP (GYPGQV) evoked spike discharge in 79 or 23% of myenteric neurons, respectively. The PAR1-AP response was mimicked by the endogenous PAR1 activator thrombin and blocked by the PAR1 antagonists SCH79797. Human myenteric neurons did not respond to PAR2-AP. This was not due to culture conditions because all three PAR-APs evoked action potentials in cultured guinea pig myenteric neurons. Consecutive application of PAR-APs revealed coexpression (relative to the population responding to PAR-APs) of PAR1/PAR2 in 51%, PAR1/PAR4 in 43%, and of PAR2/PAR4 in 29% of guinea pig myenteric neurons. Our study provided further evidence for the prominent role of neuronal PAR1 in the human enteric nervous system. PMID:22988431

  6. In Vitro Infection of Trypanosoma cruzi Causes Decrease in Glucose Transporter Protein-1 (GLUT1) Expression in Explants of Human Placental Villi Cultured under Normal and High Glucose Concentrations

    PubMed Central

    Mezzano, Luciana; Repossi, Gastón; Fretes, Ricardo E.; Lin, Susana; Sartori, María José; de Fabro, Sofía G. Parisi

    2012-01-01

    Trypanosoma cruzi, the etiologic Chagas' disease agent, induces changes in protein pattern of the human placenta syncytiotrophoblast. The glucose transporter protein-1 (GLUT1) is the primary isoform involved in transplacental glucose transport. We carried out in vitro assays to determine if T. cruzi infection would induce changes in placental GLUT1 protein expression under normal and high concentration of glucose. Using Western blot and immunohistological techniques, GLUT1 expression was determined in normal placental villi cultured under normal or high concentrations of glucose, with or without in vitro T. cruzi infection, for 24 and 48 hours. High glucose media or T. cruzi infection alone reduced GLUT1 expression. A yet more accentuated reduction was observed when infection and high glucose condition took place together. We inform, for the first time, that T. cruzi infection may induce reduction of GLUT1 expression under normal and high glucose concentrations, and this effect is synergic to high glucose concentrations. PMID:21941569

  7. In Vitro Infection of Trypanosoma cruzi Causes Decrease in Glucose Transporter Protein-1 (GLUT1) Expression in Explants of Human Placental Villi Cultured under Normal and High Glucose Concentrations.

    PubMed

    Mezzano, Luciana; Repossi, Gastón; Fretes, Ricardo E; Lin, Susana; Sartori, María José; de Fabro, Sofía G Parisi

    2012-01-01

    Trypanosoma cruzi, the etiologic Chagas' disease agent, induces changes in protein pattern of the human placenta syncytiotrophoblast. The glucose transporter protein-1 (GLUT1) is the primary isoform involved in transplacental glucose transport. We carried out in vitro assays to determine if T. cruzi infection would induce changes in placental GLUT1 protein expression under normal and high concentration of glucose. Using Western blot and immunohistological techniques, GLUT1 expression was determined in normal placental villi cultured under normal or high concentrations of glucose, with or without in vitro T. cruzi infection, for 24 and 48 hours. High glucose media or T. cruzi infection alone reduced GLUT1 expression. A yet more accentuated reduction was observed when infection and high glucose condition took place together. We inform, for the first time, that T. cruzi infection may induce reduction of GLUT1 expression under normal and high glucose concentrations, and this effect is synergic to high glucose concentrations.

  8. Analysis of cell identity, morphology, apoptosis and mitotic activity in a primary neural cell culture system in Drosophila

    PubMed Central

    2012-01-01

    In Drosophila, most neurogenetic research is carried out in vivo. Mammalian research demonstrates that primary cell culture techniques provide a powerful model to address cell autonomous and non-autonomous processes outside their endogenous environment. We developed a cell culture system in Drosophila using wildtype and genetically manipulated primary neural tissue for long-term observations. We assessed the molecular identity of distinct neural cell types by immunolabeling and genetically expressed fluorescent cell markers. We monitored mitotic activity of cell cultures derived from wildtype and tumorous larval brains. Our system provides a powerful approach to unveil developmental processes in the nervous system and to complement studies in vivo. PMID:22554060

  9. Characterization of rat parotid and submandibular acinar cell apoptosis in primary culture.

    PubMed

    Limesand, Kirsten H; Barzen, Katherine A; Sanders, Linda A; Sclafani, Robert A; Raynolds, Mary V; Reyland, Mary E; Anderson, Steven M; Quissell, David O

    2003-01-01

    Apoptosis is a highly organized cellular process that is critical for maintaining glandular homeostasis. We have used primary rat salivary acinar cells from the parotid and submandibular glands to investigate the critical regulatory events involved in apoptosis. Caspase-3 activity, cleavage of caspase substrates, and deoxyribonucleic acid (DNA) fragmentation were assayed in cells treated with etoposide, a DNA-damaging agent, or brefeldin A (BFA), a Golgi toxin. Dose-response studies showed that the sensitivity of both cell types to etoposide and BFA was similar, with 150 microM etoposide or 1.5 microM BFA inducing maximal caspase activation. However, BFA induced a more robust activation of caspase and DNA fragmentation in both cell types. Similar results were observed when the caspase cleavage of poly(adenosine 5'-diphosphate ribose) polymerase and protein kinase C delta were analyzed by Western blot. Analysis of the kinetics of apoptosis showed that caspase-3 activation was maximal at 8 h of etoposide or BFA treatment in the parotid cells and at 8-18 h in the submandibular cells. A similar time course was observed when DNA fragmentation was assayed, although maximal DNA fragmentation in BFA-treated cells was two- to threefold higher than that observed in etoposide-treated cells. Despite slight kinetic differences, it would appear that the apoptotic cascade is very similar in both primary parotid and submandibular acinar cells. Although limited in their long-term stability in culture, the use of primary, nonimmortalized salivary acinar cultures will also permit the use of specific transgenic animals to further characterize the molecular events involved in the regulation of salivary gland acinar cell apoptosis.

  10. Wnt3a induces exosome secretion from primary cultured rat microglia

    PubMed Central

    2012-01-01

    Background Microglia, the immune effector cells of the CNS and the signaling molecule Wnt, both play critical roles in neurodevelopment and neurological disease. Here we describe the inducible release of exosomes from primary cultured rat microglia following treatment with recombinant carrier-free Wnt3a. Results Wnt3a was internalised into microglia, being detectable in early endosomes, and secreted in exosomes through a GSK3-independent mechanism. Electron microscopy demonstrated that exosomes were elliptical, electron-dense (100 nm) vesicles that coalesced with time in vitro. In contrast to microglia, primary cortical neurons released exosomes constitutively and the quantity of exosomes released was not altered by Wnt3a treatment. The proteomic profile of the microglial-derived exosomes was characterised using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and the vesicles were found to be associated with proteins involved in cellular architecture, metabolism, protein synthesis and protein degradation including β-actin, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunits and ubiquitin (45 proteins in total). Unlike lipopolysaccharide, Wnt3a did not induce a neurotoxic, pro-inflammatory phenotype in primary microglia. Conclusion These findings reveal a novel mechanism through which Wnt3a signals in microglia resulting in the release of exosomes loaded with proteinaceous cargo. PMID:23173708

  11. Differential Proteomic Analysis of Human Placenta-Derived Mesenchymal Stem Cells Cultured on Normal Tissue Culture Surface and Hyaluronan-Coated Surface

    PubMed Central

    Wong, Tzyy Yue; Chen, Ying-Hui; Liu, Szu-Heng; Solis, Mairim Alexandra; Yu, Chen-Hsiang; Chang, Chiung-Hsin; Huang, Lynn L. H.

    2016-01-01

    Our previous results showed that hyaluronan (HA) preserved human placenta-derived mesenchymal stem cells (PDMSC) in a slow cell cycling mode similar to quiescence, the pristine state of stem cells in vivo, and HA was found to prevent murine adipose-derived mesenchymal stem cells from senescence. Here, stable isotope labeling by amino acid in cell culture (SILAC) proteomic profiling was used to evaluate the effects of HA on aging phenomenon in stem cells, comparing (1) old and young passage PDMSC cultured on normal tissue culture surface (TCS); (2) old passage on HA-coated surface (CHA) compared to TCS; (3) old and young passage on CHA. The results indicated that senescence-associated protein transgelin (TAGLN) was upregulated in old TCS. Protein CYR61, reportedly senescence-related, was downregulated in old CHA compared to old TCS. The SIRT1-interacting Nicotinamide phosphoribosyltransferase (NAMPT) increased by 2.23-fold in old CHA compared to old TCS, and is 0.48-fold lower in old TCS compared to young TCS. Results also indicated that components of endoplasmic reticulum associated degradation (ERAD) pathway were upregulated in old CHA compared to old TCS cells, potentially for overcoming stress to maintain cell function and suppress senescence. Our data points to pathways that may be targeted by HA to maintain stem cells youth. PMID:27057169

  12. Development of primary cell cultures using hemocytes and phagocytic tissue cells of Locusta migratoria: an application for locust immunity studies.

    PubMed

    Duressa, Tewodros Firdissa; Huybrechts, Roger

    2016-01-01

    Insect cell cultures played central roles in unraveling many insect physiological and immunological processes. Regardless, despite imminent needs, insect cell lines were developed primarily from Dipteran and Lepidopteran orders, leaving many important insects such as Orthopteran locusts under-represented. Besides the lack of cell lines, the slow progress in development of in vitro techniques is attributed to poor communications between different laboratories regarding optimized primary cell cultures. Therefore, we report here about methods developed for primary cell culture of Locusta migratoria hemocyte and phagocytic tissue cells by which we could maintain viable hemocytes in vitro for over 5 d and phagocytic tissue cells for over 12 d. 2-Mercaptoethanol and phenyl-thiourea supplements in Grace's medium together with addition of fetal bovine serum 30 min after cell seeding resulted in a successful setup of the primary cell cultures and a week-long survival of the hemocytes and phagocytic tissue cells in vitro.

  13. Primary cultures of renal epithelial cells from X-linked hypophosphatemic (Hyp) mice express defects in phosphate transport and vitamin D metabolism.

    PubMed Central

    Bell, C L; Tenenhouse, H S; Scriver, C R

    1988-01-01

    Mutation in a gene (symbol Hyp) on the X chromosome causes hypophosphatemia in the mouse. The murine phenotype is a counterpart of X-linked hypophosphatemia in man. Both exhibit impaired renal reabsorption of phosphate in vivo. In vitro studies in the Hyp mouse have shown decreased Na+-dependent phosphate transport at the brush border membrane and abnormal mitochondrial vitamin D metabolism. To determine whether the mutant renal phenotype is intrinsic to the kidney or dependent upon putative extrinsic humoral factor(s) for its expression, we established primary cultures of renal epithelial cells from normal and Hyp male mouse kidneys. The cells are derived from proximal tubule. Initial uptake rates of phosphate and alpha-methyl-D-glucopyranoside (alpha-MG), a metabolically inert analogue of D-glucose, were measured simultaneously in confluent monolayers exhibiting epithelial polarity and tight junctions. The mean phosphate/alpha-MG uptake ratio in Hyp cultures was 82% of that in normal cells (P less than 0.01, n = 96). Moreover, the production of 24,25-dihydroxyvitamin D3 was significantly elevated in confluent cultures of Hyp cells relative to normal cells. These results imply that the Hyp gene is expressed in situ in renal epithelium and suggest that humoral factors are not necessary for the mutant renal phenotype in X-linked hypophosphatemia of mouse and man. PMID:3414685

  14. Enhanced depth imaging detects lamina cribrosa thickness differences in normal tension glaucoma and primary open-angle glaucoma.

    PubMed

    Park, Hae-Young Lopilly; Jeon, So Hee; Park, Chan Kee

    2012-01-01

    To confirm the advantages of the enhanced depth imaging (EDI) mode over the standard mode of the Heidelberg Spectralis spectral domain optical coherence tomography (SD-OCT) for imaging of the lamina cribrosa, and to compare laminar thicknesses of various glaucoma types with or without disc hemorrhage in a similar state of visual field loss. Cross-sectional, case-control design. We included 137 glaucoma patients and 49 healthy controls. Optic nerve head B-scans were obtained by both the standard and EDI modes of the Spectralis OCT. Laminar thickness was measured at the center of mid-superior, central, and mid-inferior horizontal B-scans. Laminar thickness in patients with normal tension glaucoma (NTG) was compared with that in patients with primary open-angle glaucoma (POAG). To verify the reproducibility of EDI imaging, intraclass correlation coefficients and test-retest variability were calculated from selected B-scans. Laminar thickness and mean deviation values on standard automatic perimetry. The EDI OCT imaging showed significantly better intraobserver, interobserver, intravisit, and intervisit reproducibility than those by standard imaging. Laminar thickness in mid-superior, central, and mid-inferior regions was thinner in the POAG and NTG groups than in the normal control group (P<0.001). The mid-superior, central, and mid-inferior regions of the lamina were also significantly thinner in patients with NTG and disc hemorrhage than in those with NTG but no disc hemorrhage. The EDI mode of the Heidelberg Spectralis SD-OCT detected differences in the lamina cribrosa by glaucoma type. The lamina cribrosa was thinner in NTG eyes and in NTG eyes with disc hemorrhage. The authors have no proprietary or commercial interest in any of the materials discussed in this article. Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

  15. Primary Murine Small Intestinal Epithelial Cells, Maintained in Long-Term Culture, Are Susceptible to Rotavirus Infection

    PubMed Central

    Macartney, Kristine K.; Baumgart, Daniel C.; Carding, Simon R.; Brubaker, Jeffery O.; Offit, Paul A.

    2000-01-01

    We describe a method for long-term culture of primary small intestinal epithelial cells (IEC) from suckling mice. IEC were digested from intestinal fragments as small intact units of epithelium (organoids) by using collagenase and dispase. IEC proliferated from organoids on a basement-membrane-coated culture surface and remained viable for 3 weeks. Cultured IEC had the morphologic and functional characteristics of immature enterocytes, notably sustained expression of cytokeratin and alkaline phosphatase. Few mesenchymal cells were present in the IEC cultures. IEC were also cultured from adult BALB/c mice and expressed major histocompatibility complex (MHC) class II antigens for at least 48 h in vitro. Primary IEC supported the growth of rhesus rotavirus (RRV) to a greater extent than a murine small intestinal cell line, m-ICcl2. Cell-culture-adapted murine rotavirus strain EDIM infected primary IEC and m-ICcl2 cells to a lesser extent than RRV. Wild-type EDIM did not infect either cell type. Long-term culture of primary murine small intestinal epithelial cells provides a method to study (i) virus-cell interactions, (ii) the capacity of IEC to act as antigen-presenting cells using a wide variety of MHC haplotypes, and (iii) IEC biology. PMID:10823867

  16. Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes

    SciTech Connect

    Ruch, R.J.; Klaunig, J.E.; Schultz, N.E.; Askari, A.B.; Lacher, D.A.; Pereira, M.A.; Goldblatt, P.J.

    1986-11-01

    Mechanisms of chloroform (CHCl/sub 3/) and carbon tetrachloride (CCl/sub 4/) toxicity to primary cultured male B6C3F1 mouse hepatocytes were investigated. The cytotoxicity of both CHCl/sub 3/ and CCl/sub 4/ was dose- and duration-dependent. Maximal hepatocyte toxicity, as determined by lactate dehydrogenase leakage into the culture medium, occurred with the highest concentrations of CHCl/sub 3/ (5 mM) and CCl/sub 4/ (2.5 mM) used and with the longest duration of treatment (20 hr). CCl/sub 4/ was approximately 16 times more toxic than CHCl/sub 3/ to the hepatocytes. The toxicity of these compounds was decreased by adding the mixed function oxidase system (MFOS) inhibitor, SKF-525A (25..mu..M) to the cultures. The addition of diethyl maleate (0.25 mM), which depletes intracellular glutathione (GSH)-potentiated CHCl/sub 3/ and CCl/sub 4/ toxicity. The toxicity of CHCl/sub 3/ and CCl/sub 4/ could also be decreased by adding the antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) (25..mu..M), ..cap alpha..-tocopherol acetate (Vitamin E) (0.1 mM), or superoxide dismutase (SOD) (100 U/mL) to the cultures. These results suggest that: in mouse hepatocytes, both CHCl/sub 3/ and CCl/sub 4/ are metabolized to toxic components by the MFOS; GSH plays a role in detoxifying those metabolites; free radicals are produced during the metabolism of CHCl/sub 3/ and CCl/sub 4/; and free radicals may be important mediators of the toxicity of these two halomethanes.

  17. A primary culture system of adult rat heart cells for the evaluation of cocaine toxicity.

    PubMed

    Welder, A A

    1992-01-01

    The complex dose-response relationship by which cocaine (Coc) directly precipitates unfavorable cardiac consequences are not known. There appears to be two diametrically opposed cardiovascular actions of Coc. At low doses, the sympathetic nervous system responses dominate, whereas, at high doses, the local anesthetic actions exert the most powerful effects. The purpose of this study was to describe a dose- and time-dependent Coc cardiotoxicity profile in a model of spontaneously contracting adult primary myocardial cell cultures obtained from 60-90-day-old Sprague-Dawley rats. Indices of toxicity determined included contractility, morphology, lactate dehydrogenase release (LDH), mitochondrial tetrazolium formazan (MTT) production and neutral red (NR) formation. After the cells had been grown in culture for 11 days, they were exposed to 1 x 10(-3), 1 x 10(-5), 1 x 10(-7) and 1 x 10(-9) M Coc for 1-24 h. The two lowest doses of Coc (1 x 10(-7) and 1 x 10(-9) M) had little or no effect on the adult heart cell cultures. However, morphological alterations included vacuolization, granulation and pseudopodia formation as early as 1 h after exposure to the highest doses of Coc (1 x 10(-3) and 1 x 10(-5) M). For all time points observed, the two highest doses of Coc (1 x 10(-3) and 1 x 10(-5) M) significantly depressed contractility and induced significant LDH release. MTT formazan production and NR retention were not significantly different from untreated controls for all treatments. By employing an acute Coc exposure paradigm, these data demonstrate that Coc doses greater than or equal to 1 x 10(-5) M induce direct injurious local anesthetic effects on contractility and morphology of spontaneously contracting adult rat myocardial cells in culture.

  18. Evaluation of primary and secondary production using wastewater as a culture medium.

    PubMed

    Nandini, S; Ramírez-García, Pedro; Sarma, S S S

    2010-10-01

    The ability of rotifers and cladocerans to convert primary to secondary production in wastewaters was tested. Scenedesmus acutus was cultured on Bold's (defined) medium, wastewater from the tertiary phase of water treatment and a mixture of both. The algal growth rates (µ) ranged from 0.4 to 0.7 day⁻¹, being highest in defined medium. The demographic characteristics of Brachionus rubens and Moina macrocopa were tested using algae at a density of 1.0 x 10⁶ cells mL⁻¹. Into each test jar, we introduced 20 neonates (< 12-h-old) of either B. rubens or M. macrocopa. Daily (for M. macrocopa) or twice a day (for B. rubens), dead adults and the neonates were enumerated and removed. Average life-span and generation time of B. rubens were not significantly influenced by the algal treatment type. Gross and net reproductive rates were significantly influenced by the medium on which the algae was cultured; in the case of B. rubens, they ranged from 20-36 and 10-22 offspring female⁻¹; the corresponding values for M. macrocopa were higher (38-110 and 13-31 offspring female⁻¹, respectively). The rate of population increase was higher for Brachionus (0.41-0.65 day⁻¹)) compared to Moina (0.28-0.57 day⁻¹). Brachionus had significantly higher growth rates on algae cultured on Bold medium than on treated wastewater while Moina grew significantly better on Scenedesmus cultured on Bold medium or a mixture of treated wastewater and Bold medium than on treated wastewater alone.

  19. Steatotic rat hepatocytes in primary culture are more susceptible to the acute toxic effect of acetaminophen.

    PubMed

    Kučera, O; Al-Dury, S; Lotková, H; Roušar, T; Rychtrmoc, D; Červinková, Z

    2012-01-01

    Acetaminophen (APAP) overdose is the most common cause of acute liver failure in humans. Non-alcoholic fatty liver disease is the most frequent chronic liver disease in developed countries. The aim of our work was to compare the effect of APAP on intact rat hepatocytes and hepatocytes isolated from steatotic liver in primary cultures. Male Wistar rats were fed with standard diet (10 % energy from fat) and high-fat diet (71 % energy from fat) for 6 weeks and then hepatocytes were isolated. After cell attachment, APAP (1; 2.5; 3.75 and 5 mM) was added to culture media (William's E medium) and hepatocytes were cultured for up to 24 hours. APAP caused more severe dose-dependent damage of steatotic hepatocytes as documented by increased release of lactate dehydrogenase (LDH) and LDH leakage, decreased activity of cellular dehydrogenases (WST-1 test) and reduced albumin production. Intact steatotic hepatocytes contained lower amount of reduced glutathione (GSH). Treatment with APAP (1 and 2.5 mmol/l) caused more pronounced decrease in GSH in steatotic hepatocytes. ROS (reactive oxygen species) formation after 24-hour incubation was significantly higher in fatty hepatocytes using APAP at concentration of 3.75 and 5 mmol/l. Interleukin 6 (IL-6) production was elevated in 2.5 mM APAP-treated nonsteatotic and steatotic hepatocyte cultures at 8 hours, compared to appropriate controls. In conclusions, our results indicate that steatotic hepatocytes exert higher sensitivity to the toxic action of APAP. This sensitivity may be caused by lower content of GSH in intact steatotic hepatocytes and by more pronounced APAP-induced decrease in intracellular concentration of GSH.

  20. Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes.

    PubMed Central

    Ruch, R J; Klaunig, J E; Schultz, N E; Askari, A B; Lacher, D A; Pereira, M A; Goldblatt, P J

    1986-01-01

    Mechanisms of chloroform (CHCl3) and carbon tetrachloride (CCl4) toxicity to primary cultured male B6C3F1 mouse hepatocytes were investigated. The cytotoxicity of both CHCl3 and CCl4 was dose- and duration-dependent. Maximal hepatocyte toxicity, as determined by lactate dehydrogenase leakage into the culture medium, occurred with the highest concentrations of CHCl3 (5 mM) and CCl4 (2.5 mM) used and with the longest duration of treatment (20 hr). CCl4 was approximately 16 times more toxic than CHCl3 to the hepatocytes. The toxicity of these compounds was decreased by adding the mixed function oxidase system (MFOS) inhibitor, SKF-525A (25 microM) to the cultures. The addition of diethyl maleate (0.25 mM), which depletes intracellular glutathione (GSH)-potentiated CHCl3 and CCl4 toxicity. The toxicity of CHCl3 and CCl4 could also be decreased by adding the antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) (25 microM), alpha-tocopherol acetate (Vitamin E) (0.1 mM), or superoxide dismutase (SOD) (100 U/mL) to the cultures. These results suggest that: in mouse hepatocytes, both CHCl3 and CCl4 are metabolized to toxic components by the MFOS; GSH plays a role in detoxifying those metabolites; free radicals are produced during the metabolism of CHCl3 and CCl4; and free radicals may be important mediators of the toxicity of these two halomethanes. PMID:3816733

  1. Excitatory amino acid receptor-stimulated phosphoinositide turnover in primary cerebrocortical cultures.

    PubMed Central

    Birrell, G. J.; Marcoux, F. W.

    1993-01-01

    1. Characterization of excitatory amino acid-induced accumulation of [3H]-phosphoinositides was carried out in primary cerebrocortical cultures isolated from foetal rats. 2. All of the excitatory amino acid receptor agonists examined caused concentration-dependent enhancement of phosphoinositide (PI) formation. The most potent excitatory amino acid receptor agonists were quisqualate, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD), ibotenate and glutamate with mean EC50 values of 0.9 +/- 0.4 microM, 15 +/- 5 microM, 15 +/- 3 microM and 41 +/- 8 microM respectively. 3. The selective ionotropic receptor antagonists kynurenic acid (1 mM), 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX, 10 microM) and (+/-)-4-(3-phosphonopropyl)-2 piperazinecarboxylic acid (CPP, 100 microM), failed to block responses to quisqualate, (1S,3R)-ACPD or glutamate. D,L-2-Amino-3-phosphonopropionate (D,L-AP3) did not block 1S,3R-ACPD or quisqualate-induced PI turnover, but had an additive effect with quisqualate or (1S,3R)-ACPD. 4. Exposure of cultures to agonists in the absence of added extracellular calcium reduced the maximal quisqualate response by approximately 45%, revealing a two-component concentration-response curve. Concentration-response curves to ibotenate and glutamate became flattened by omission of extracellular calcium, whereas (1S,3R)-ACPD-stimulated PI turnover was unaffected. 5. Pretreatment of cultures with pertussis toxin markedly inhibited PI responses evoked by (1S,3R)-ACPD. 6. These results suggest that excitatory amino acid-stimulated PI turnover in cerebrocortical cultures is independent of ionotropic receptor activation and is mediated via specific G-protein-linked metabotropic receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8395285

  2. Comparative Analysis of Human and Rodent Brain Primary Neuronal Culture Spontaneous Activity Using Micro-Electrode Array Technology.

    PubMed

    Napoli, Alessandro; Obeid, Iyad

    2016-03-01

    Electrical activity in embryonic brain tissue has typically been studied using Micro Electrode Array (MEA) technology to make dozens of simultaneous recordings from dissociated neuronal cultures, brain stem cell progenitors, or brain slices from fetal rodents. Although these rodent neuronal primary culture electrical properties are mostly investigated, it has not been yet established to what extent the electrical characteristics of rodent brain neuronal cultures can be generalized to those of humans. A direct comparison of spontaneous spiking activity between rodent and human primary neurons grown under the same in vitro conditions using MEA technology has never been carried out before and will be described in the present study. Human and rodent dissociated fetal brain neuronal cultures were established in-vitro by culturing on a glass grid of 60 planar microelectrodes neurons under identical conditions. Three different cultures of human neurons were produced from tissue sourced from a single aborted fetus (at 16-18 gestational weeks) and these were compared with seven different cultures of embryonic rat neurons (at 18 gestational days) originally isolated from a single rat. The results show that the human and rodent cultures behaved significantly differently. Whereas the rodent cultures demonstrated robust spontaneous activation and network activity after only 10 days, the human cultures required nearly 40 days to achieve a substantially weaker level of electrical function. These results suggest that rat neuron preparations may yield inferences that do not necessarily transfer to humans.

  3. Isolation, characterization, and expansion methods for defined primary renal cell populations from rodent, canine, and human normal and diseased kidneys.

    PubMed

    Presnell, Sharon C; Bruce, Andrew T; Wallace, Shay M; Choudhury, Sumana; Genheimer, Christopher W; Cox, Bryan; Guthrie, Kelly; Werdin, Eric S; Tatsumi-Ficht, Patricia; Ilagan, Roger M; Kelley, Russell W; Rivera, Elias A; Ludlow, John W; Wagner, Belinda J; Jayo, Manuel J; Bertram, Timothy A

    2011-03-01

    Chronic kidney disease (CKD) is a global health problem; the growing gap between the number of patients awaiting transplant and organs actually transplanted highlights the need for new treatments to restore renal function. Regenerative medicine is a promising approach from which treatments for organ-level disorders (e.g., neurogenic bladder) have emerged and translated to clinics. Regenerative templates, composed of biodegradable material and autologous cells, isolated and expanded ex vivo, stimulate native-like organ tissue regeneration after implantation. A critical step for extending this strategy from bladder to kidney is the ability to isolate, characterize, and expand functional renal cells with therapeutic potential from diseased tissue. In this study, we developed methods that yield distinct subpopulations of primary kidney cells that are compatible with process development and scale-up. These methods were translated to rodent, large mammal, and human kidneys, and then to rodent and human tissues with advanced CKD. Comparative in vitro studies demonstrated that phenotype and key functional attributes were retained consistently in ex vivo cultures regardless of species or disease state, suggesting that autologous sourcing of cells that contribute to in situ kidney regeneration after injury is feasible, even with biopsies from patients with advanced CKD.

  4. Biosynthesis of normal and low-molecular-mass complement component C1q by cultured human monocytes and macrophages.

    PubMed Central

    Hoekzema, R; Brouwer, M C; de Graeff-Meeder, E R; van Helden, H P; Hack, C E

    1989-01-01

    High levels of low-molecular-mass complement component C1q (LMM-C1q), a haemolytically inactive form of C1q, are found in serum of individuals with inherited complete (functional) C1q deficiency and in serum of patients with systemic lupus erythematosus, whereas lower levels are present in normal serum [Hoekzema, Hannema, Swaak, Paardekooper & Hack (1985) J. Immunol. 135, 265-271]. To investigate whether LMM-C1q is a (by-)product of C1q synthesis or the result of degradation of C1q, cultures of blood monocytes and of alveolar macrophages, which secrete functional C1q, were studied. A considerable portion of C1q-like protein secreted by these cells was found to be LMM-C1q. In contrast with the C1q fragments that resulted from degradation of normal C1q during phagocytosis, culture-derived LMM-C1q appeared to be identical with LMM-C1q found in serum, as judged by sedimentation behaviour, subunit structure and recognition by poly- and mono-clonal antibodies raised against C1q. The presence of LMM-C1q in cytoplasmic organelles compatible with the Golgi apparatus and the inability to generate LMM-C1q by impeding hydroxylation and triple-helix formation of C1q further argues against degradation as its source. Monocyte cultures of homozygous probands from two families with complete functional C1q deficiency reflected the abnormalities in serum, i.e. absence of functional C1q, but increased levels of LMM-C1q. By contrast, secretion of C1q and LMM-C1q by cells from healthy individuals was clearly co-ordinate, indicating that LMM-C1q in serum may provide a unique marker of C1q synthesis in vivo. Images Fig. 1. Fig. 3. Fig. 4. Fig. 6. PMID:2649076

  5. Primary transitions between the yrast superdeformed band and low-lying normal deformed states in {sup 194}Pb

    SciTech Connect

    Hauschild, K.; Bernstein, L.A.; Becker, J.A.

    1996-12-31

    The observation of one-step `primary` gamma-ray transitions directly linking the superdeformed (SD) states to the normal deformed (ND) low-lying states of known excitation energies (E{sub x}), spins and parities (J{sup {pi}}) is crucial to determining the E{sub x} and J{sup {pi}} of the SD states. With this knowledge one can begin to address some of the outstanding problems associated with SD nuclei, such as the identical band issue, and one can also place more stringent restrictions on theoretical calculations which predict SD states and their properties. Brinkman, et al., used the early implementation of the GAMMASPHERE spectrometer array (32 detectors) and proposed a single, candidate {gamma} ray linking the {sup 194}Pb yrast SD band to the low-lying ND states in {sup 194}Pb. Using 55 detectors in the GAMMASPHERE array Khoo, et al., observed multiple links between the yrast SD band in {sup 194}Hg and the low-lying level scheme and conclusively determined E{sub x} and J of the yrast SD states. Here the authors report on an experiment in which Gammasphere with 88 detectors was used and the E{sub x} and J{sup {pi}} values of the yrast SD states in {sup 194}Pb were uniquely determined. Twelve one-step linking transitions between the yrast SD band and low-lying states in {sup 194}Pb have been identified, including the transition proposed by Brinkman. These transitions have been placed in the level scheme of {sup 194}Pb using coincidence relationships and agreements between the energies of the primary transitions and the energy differences in level spacings. Furthermore, measurements of angular asymmetries have yielded the multipolarities of the primaries which have allowed J{sup {pi}} assignments of the {sup 194}Pb SD states to be unambiguously determined for the first time without a priori assumptions about the character of SD bands. A study performed in parallel to this work using the EUROGAM-II array reports similar, but somewhat less extensive, results.

  6. Propagation of normal human epithelial cell populations using an in vivo culture system. Description and applications.

    PubMed Central

    Klein-Szanto, A. J.; Terzaghi, M.; Mirkin, L. D.; Martin, D.; Shiba, M.

    1982-01-01

    A new model using xenotransplanted human epithelia was developed for the study of toxic and carcinogenic effects of chemicals. Epithelial cells from the respiratory tract of 4 male and 3 female premature and fullterm fetuses were enzymatically removed and inoculated into deepithelialized rat tracheas. These were sealed at both ends and transplanted subcutaneously into nude mice. After 3-4 weeks, a normal mucociliary epithelium covered the tracheal lumen. At this stage the epithelial cells could be isolated again and transplanted into new denuded rat tracheas. This passaging could be repeated up to six times, each permitting an amplification factor of approximately 3. Tracheal transplants containing cells of human origin (in vivo Passages 2-4) were treated with 7,12-dimethylbenz(a)anthracene. Hyperplasias, squamous metaplasias, and dysplasias were seen 1-8 weeks after initiation of treatment, indicating that the responses of human and rodent epithelial cells to polycyclic aromatic hydrocarbons are similar. Initial experiments with skin and esophageal epithelia suggest that other covering epithelia could also be used in this fashion for evaluation of toxicants and carcinogens that are likely to come into contact with these tissues. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:6821529

  7. Development and validation of primary human myometrial cell culture models to study pregnancy and labour.

    PubMed

    Mosher, Andrea A; Rainey, Kelly J; Bolstad, Seunghwa S; Lye, Stephen J; Mitchell, Bryan F; Olson, David M; Wood, Stephen L; Slater, Donna M

    2013-01-01

    The development of the in vitro cell culture model has greatly facilitated the ability to study gene expression and regulation within human tissues. Within the human uterus, the upper (fundal) segment and the lower segment may provide distinct functions throughout pregnancy and during labour. We have established primary cultured human myometrial cells, isolated from both upper and lower segment regions of the pregnant human uterus, and validated them for the purpose of studying human pregnancy and labour. The specific objectives of this study were to monitor the viability and characterize the expression profile using selected cellular, contractile and pregnancy associated markers in the primary cultured human myometrial cells. Labour has been described as an inflammatory process; therefore, the ability of these cells to respond to an inflammatory stimulus was also investigated. Myometrial cells isolated from paired upper segment (US) and lower segment (LS) biopsies, obtained from women undergoing Caesarean section deliveries at term prior to the onset of labour, were used to identify expression of; α smooth muscle actin, calponin, caldesmon, connexin 43, cyclo-oxygenase-2 (COX-2), oxytocin receptor, tropomyosin and vimentin, by RT-PCR and/or immunocytochemistry. Interleukin (IL)-1β was used to treat cells, subsequently expression of COX-2 mRNA and release of interleukin-8 (CXCL8), were measured. ANOVA followed by Bonferroni's multiple comparisons test was performed. We demonstrate that US and LS human myometrial cells stably express all markers examined to at least passage ten (p10). Connexin 43, COX-2 and vimentin mRNA expression were significantly higher in LS cells compared to US cells. Both cell populations respond to IL-1β, demonstrated by a robust release of CXCL8 and increased expression of COX-2 mRNA from passage one (p1) through to p10. Isolated primary myometrial cells maintain expression of smooth muscle and pregnancy-associated markers and retain

  8. Thyroid hormones are needed to sustain "inappropriately" normal TSH during non-thyroidal illness syndrome: a clinical observation in severely ill patients with primary hypothyroidism.

    PubMed

    Lado-Abeal, Joaquin

    2015-01-01

    Non-thyroidal illness syndrome (NTIS) is a form of hypothyroidism that occurs during illness. NTIS is characterized by low thyroid hormones (TH) level in the serum and tissues, with normal serum TSH levels that are considered "inappropriately" normal. The mechanisms responsible for a lack of increase in serum TSH during NTIS are unknown. However, a decreased expression of hypothalamic TRH has been documented in deceased humans with NTIS. It has been suggested that pro-inflammatory cytokines are responsible for the inhibition of TRH release. If a similar TSH response occurs in severely ill patients with primary hypothyroidism has not been reported. Seven severely ill patients with unknown or poorly treated primary hypothyroidism are presented. Serum TSH and TH were measured at admission and during TH replacement. Tertiary university hospital. Besides suffering severe illness, patient's serum TSH was high and decreased once TH replacement was given. Levothyroxine administration was enough to down regulate TSH secretion. Serum reverse T3 (rT3) was normal or high and showed further elevation with stepwise increase in levothyroxine doses. The thyroid axis inhibitory feedback system is fully preserved in primary hypothyroidism during severe illness. In NTIS, serum TSH levels remain within normal limits because the supply of TH to TRH neurons is sustained normally. Pro-inflammatory cytokines does not play a major role inhibiting TRH release during NTIS associated to primary hypothyroidism. D3 activity increases in humans with severe illness, a mechanism behind the increased requirements in TH replacement to achieve normal serum TH.

  9. Determining the Long-term Effect of Antibiotic Administration on the Human Normal Intestinal Microbiota Using Culture and Pyrosequencing Methods.

    PubMed

    Rashid, Mamun-Ur; Zaura, Egijia; Buijs, Mark J; Keijser, Bart J F; Crielaard, Wim; Nord, Carl Erik; Weintraub, Andrej

    2015-05-15

    The purpose of the study was to assess the effect of ciprofloxacin (500 mg twice daily for 10 days) or clindamycin (150 mg 4 times daily for 10 days) on the fecal microbiota of healthy humans for a period of 1 year as compared to placebo. Two different methods, culture and microbiome analysis, were used. Fecal samples were collected for analyses at 6 time-points. The interval needed for the normal microbiota to be normalized after ciprofloxacin or clindamycin treatment differed for various bacterial species. It took 1-12 months to normalize the human microbiota after antibiotic administration, with the most pronounced effect on day 11. Exposure to ciprofloxacin or clindamycin had a strong effect on the diversity of the microbiome, and changes in microbial composition were observed until the 12th month, with the most pronounced microbial shift at month 1. No Clostridium difficile colonization or C. difficile infections were reported. Based on the pyrosequencing results, it appears that clindamycin has more impact than ciprofloxacin on the intestinal microbiota. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Anisotonic media and glutamate-induced ion transport and volume responses in primary astrocyte cultures.

    PubMed

    Kimelberg, H K

    1987-01-01

    1. The responses of primary monolayer astrocyte cultures prepared from neonatal rat brains to hyper- and hypotonic media and to the addition of L-glutamic acid were examined as part of a systematic approach to use these cultures to obtain information on the mechanisms of the volume changes seen in astroglial cells in situ. 2. Addition of 200 mM mannitol to the medium to make it hypertonic caused cell shrinkage as measured with [14C]3-O-methyl-D-glucose, and also activated K+ and Cl- uptake measured with 86Rb+ and 36Cl- respectively. The increased ion uptake was completely inhibited by 0.1 mM bumetanide, showing that the Na+ + K+ + 2 Cl- co-transport system was being activated by cell shrinkage. 3. Studies of 86Rb+ uptake as a function of external K+ and hypertonic media showed a complex pattern. Increased bumetanide-sensitive, hypertonic-stimulated uptake of 86Rb+ was seen up to 20 mM K+0, with maximum stimulation being first reached at around 2 to 5 mM K+. At concentrations greater than 20 mM K+0 there was a further increase in bumetanide-sensitive 86Rb+ uptake, but there was no stimulation of this uptake by hypertonicity. There were also increases in bumetanide-insensitive 86Rb+ fluxes at [K+]0 higher than 20 mM that may have been due to opening of voltage-dependent K+ channels; this increased 86Rb+ flux was decreased in hypertonic medium. 4. When primary astrocyte cultures were swollen in hypotonic medium there was a rapid increase in volume as measured with [14C] 3-O-methyl-D-glucose, which then decreased in the continued presence of hypotonic medium. Thus, these cells exhibit volume regulatory decrease or RVD, as described for other cells. The possible ionic bases of this phenomenon have not yet been fully examined but the initial RVD did not appear to stimulate a furosemide-sensitive cotransport system. 5. Glutamate has been implicated as a possible endogenous effector of volume change in astrocytes. In the presence of ouabain, L-glutamate led to swelling of

  11. TEAD transcription factors are required for normal primary myoblast differentiation in vitro and muscle regeneration in vivo.

    PubMed

    Joshi, Shilpy; Davidson, Guillaume; Le Gras, Stéphanie; Watanabe, Shuichi; Braun, Thomas; Mengus, Gabrielle; Davidson, Irwin

    2017-02-01

    The TEAD family of transcription factors (TEAD1-4) bind the MCAT element in the regulatory elements of both growth promoting and myogenic differentiation genes. Defining TEAD transcription factor function in myogenesis has proved elusive due to overlapping expression of family members and their functional redundancy. We show that silencing of either Tead1, Tead2 or Tead4 did not effect primary myoblast (PM) differentiation, but that their simultaneous knockdown strongly impaired differentiation. In contrast, Tead1 or Tead4 silencing impaired C2C12 differentiation showing their different contributions in PMs and C2C12 cells. Chromatin immunoprecipitation identified enhancers associated with myogenic genes bound by combinations of Tead4, Myod1 or Myog. Tead4 regulated distinct gene sets in C2C12 cells and PMs involving both activation of the myogenic program and repression of growth and signaling pathways. ChIP-seq from mature mouse muscle fibres in vivo identified a set of highly transcribed muscle cell-identity genes and sites bound by Tead1 and Tead4. Although inactivation of Tead4 in mature muscle fibres caused no obvious phenotype under normal conditions, notexin-induced muscle regeneration was delayed in Tead4 mutants suggesting an important role in myogenic differentiation in vivo. By combining knockdown in cell models in vitro with Tead4 inactivation in muscle in vivo, we provide the first comprehensive description of the specific and redundant roles of Tead factors in myogenic differentiation.

  12. TEAD transcription factors are required for normal primary myoblast differentiation in vitro and muscle regeneration in vivo

    PubMed Central

    Joshi, Shilpy; Le Gras, Stéphanie; Watanabe, Shuichi; Braun, Thomas

    2017-01-01

    The TEAD family of transcription factors (TEAD1-4) bind the MCAT element in the regulatory elements of both growth promoting and myogenic differentiation genes. Defining TEAD transcription factor function in myogenesis has proved elusive due to overlapping expression of family members and their functional redundancy. We show that silencing of either Tead1, Tead2 or Tead4 did not effect primary myoblast (PM) differentiation, but that their simultaneous knockdown strongly impaired differentiation. In contrast, Tead1 or Tead4 silencing impaired C2C12 differentiation showing their different contributions in PMs and C2C12 cells. Chromatin immunoprecipitation identified enhancers associated with myogenic genes bound by combinations of Tead4, Myod1 or Myog. Tead4 regulated distinct gene sets in C2C12 cells and PMs involving both activation of the myogenic program and repression of growth and signaling pathways. ChIP-seq from mature mouse muscle fibres in vivo identified a set of highly transcribed muscle cell-identity genes and sites bound by Tead1 and Tead4. Although inactivation of Tead4 in mature muscle fibres caused no obvious phenotype under normal conditions, notexin-induced muscle regeneration was delayed in Tead4 mutants suggesting an important role in myogenic differentiation in vivo. By combining knockdown in cell models in vitro with Tead4 inactivation in muscle in vivo, we provide the first comprehensive description of the specific and redundant roles of Tead factors in myogenic differentiation. PMID:28178271

  13. Elevated Plasma Endothelin-1 Levels in Normal Tension Glaucoma and Primary Open-Angle Glaucoma: A Meta-Analysis

    PubMed Central

    Li, Shengjie; Zhang, Aiping

    2016-01-01

    Purpose. The aim of this meta-analysis was to clarify the association between the plasma endothelin-1 level and the risks of normal tension glaucoma (NTG) and primary open-angle glaucoma (POAG). Methods. Relevant publications were collected from three databases including PubMed, EMBASE, and the Web of Science through December 31, 2015. In this study, the terms “(endothelin OR ET) AND glaucoma” were searched. Review Manager 5.2 was used to process the data. Results. Seven studies (212 cases, 164 controls) were included for the NTG analysis. The mean plasma endothelin-1 level in the NTG subjects was 0.60 pg/mL (p = 0.02, 95% CI: 0.17–1.04) higher than that of the healthy controls. Six studies (160 cases, 174 controls) were included for the POAG analysis, and the endothelin-1 level was 0.63 pg/mL (p = 0.007, 95% CI: 0.12–1.15) higher in the POAG subjects than in the healthy controls. Additionally, two studies influenced the meta-analysis results regarding the association of plasma endothelin-1 with POAG by sensitivity analysis, and the probability of publication bias was low. Conclusions. The observation that NTG and POAG subjects showed significantly elevated endothelin-1 plasma concentrations suggests that a higher plasma level of endothelin-1 might increase the risk of NTG and POAG development. PMID:27965889

  14. Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    PubMed

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-07-01

    The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be

  15. [Mycrob-1000: an alternative for the rapid determination of urine culture in the primary health level].

    PubMed

    Alarcón, Rolando Contreras; Ruiz, Fernando Travieso; Tamayo, Angela Zayas; Carmona, Gloria Roura; Varela, Estrella Alvarez; Ochoa, Gilberto Tillán; Frómeta, Nardo Ramírez

    2004-01-01

    The use of equipment Mycrob-1000 in detecting urinary infections in 4 hours in a primary health care center is evaluated. Two hundred fifty eight urine samples obtained from spontaneous miction were processed; the reference method was counting of colony-forming units per urine millimeter inoculated in Petri plaque in CLED medium. The coincidence rate between both methods was 92,31, with sensitivity and specificity rates of 79,00% and 96,95% respectively. The level of sensitivity was affected by factors not directly dependent on the equipment. High values of specificity and of coincidence achieved by this equipment in relation to the reference method facilitates its use in urine culture, making possible to differentiate negative urine samples in 4 or 5 hours and to focus work and resources on positive samples.

  16. Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies.

    PubMed

    Moura, Marcos de Assis; Amendoeira, Maria Regina Reis; Barbosa, Helene Santos

    2009-09-01

    The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.

  17. Primary hepatocyte cultures as in vitro tools for toxicity testing: quo vadis?

    PubMed

    Vinken, Mathieu; Vanhaecke, Tamara; Rogiers, Vera

    2012-04-01

    Cultures of primary hepatocytes are versatile tools that can serve many in vitro toxicity testing purposes. However, they cope with dedifferentiation, a process that is already initiated during the hepatocyte isolation procedure and that is manifested as the progressive loss of functionality upon subsequent cultivation. A number of strategies to prevent dedifferentiation have been introduced over the last decades, all which aim at re-establishing the in vivo hepatocyte micro-environment in vitro, but that are of merely limited success. Recent mechanistic insight into the mechanisms that underlie hepatocyte dedifferentiation has opened new avenues for the development of novel approaches that target the actual causes of this deteriorative pro